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The Journal of Immunology, 2004, 173: 251-258.
Copyright © 2004 by The American Association of Immunologists

LIGHT Expression by Mucosal T Cells May Regulate IFN-{gamma} Expression in the Intestine1

Offer Cohavy*, Jaclyn Zhou*, Steve W. Granger{dagger}, Carl F. Ware{dagger} and Stephan R. Targan2,*

* Cedars-Sinai Inflammatory Bowel Disease Center, Los Angeles, CA 90048; and {dagger} Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TNF superfamily of cytokines play an important role in T cell activation and inflammation. Sustained expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT) (TNFSF14) causes a pathological intestinal inflammation when constitutively expressed by mouse T cells. In this study, we characterized LIGHT expression on activated human T cell subsets in vitro and demonstrated a direct proinflammatory effect on regulation of IFN-{gamma}. LIGHT was induced in memory CD45RO CD4+ T cells and by IFN-{gamma}-producing CD4+ T cells. Kinetic analysis indicated rapid induction of LIGHT by human lamina propria T cells, reaching maximal levels by 2–6 h, whereas peripheral blood or lymph node-derived T cells required 24 h. Further analysis of intestinal specimens from a 41 patient cohort by flow cytometry indicated membrane LIGHT induction to higher peak levels in lamina propria T cells from the small bowel or rectum but not colon, when compared with lymph node or peripheral blood. Independent stimulation of the LIGHT receptor, herpesvirus entry mediator, induced IFN-{gamma} production in lamina propria T cells, while blocking LIGHT inhibited CD2-dependent induction of IFN-{gamma} synthesis, indicating a role for LIGHT in the regulation of IFN-{gamma} and as a putative mediator of proinflammatory T-T interactions in the intestinal mucosa. Taken together, these findings suggest LIGHT-herpesvirus entry mediator mediated signaling as an important immune regulatory mechanism in mucosal inflammatory responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TNFR ligand family has a critical signaling role in mammalian biology, especially in the development and regulation of the immune system (1), by controlling cell death and survival decisions (2). Significant evidence indicates that altered regulation of some TNF-related cytokines is linked to autoimmune diseases (3). For instance, deficiency of the proapoptotic Fas-Fas ligand pathway leads to autoimmune proliferative disease in humans and mice (4), whereas overexpression of the mature B cell survival factor, B cell-activating factor belonging to the TNF family, is associated with lupus nephritis in humans and mice (5). In mice, transgenic expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT)3 (TNFSF14) by T cells induced severe intestinal inflammation with autoimmune-like pathology, suggesting a specific linkage of LIGHT-mediated signaling to the intestinal compartment (6, 7).

Like other TNF-related ligands, LIGHT is a type II membrane protein that forms a biologically active homotrimer (8), which can be cleaved into a soluble form (9) or exist in an intracellular form encoded by an alternate spliced mRNA, which deletes the transmembrane region and is thus not displayed on the cell surface (10). LIGHT signals via two members of the TNFR family, herpesvirus entry mediator (HVEM, TNFRSF14) (8), and lymphotoxin (LT) {beta}R (TNFRSF3) which binds the LT{alpha}{beta} heterotrimer involved in the development and organization of peripheral lymphoid tissue (11). In addition, LIGHT binds DcR3 (TNFRSF6B), a soluble receptor (12). The LT{beta}R is found on myeloid and stromal cells, whereas HVEM is expressed prominently on lymphocytes, which do not express the LT{beta}R (13). LIGHT is expressed in the lymphoid compartment by activated T cells, but also by monocytes (8), and is likely to play an important immunomodulatory role mediating stimulatory T-T interactions via HVEM, since HVEM engagement constitutes a costimulatory signal augmenting proinflammatory cytokine production and T cell proliferation (14). Genetic deficiency of mouse LIGHT gene further demonstrated the significance of LIGHT to immune regulation (15, 16, 17). LIGHT–/– mice have normal lymphoid cell development, but demonstrate a defect in CD8+ T cell response to Ag and compromised CD8+ T cell differentiation (16). In addition, inhibition of the LT{beta}R signaling pathway with a LT{beta}R-Fc chimera decoy receptor alleviated inflammatory symptoms in the CD4+CD45RBhigh T cell transfer model of colitis, suggesting a contribution from LIGHT in this CD4+ T cell-mediated pathology (6, 18). Moreover, the human LIGHT locus is closely linked to the TNF family members, CD27 ligand (CD70, TNFSF7) and 4-1BB ligand (TNFSF9), within the MHC paralogous region on chromosome 19p13.3 (10). This region on chromosome 19 has been identified as a candidate susceptibility locus for Crohn’s disease (CD) (19), providing additional circumstantial evidence of a role for LIGHT in intestinal inflammatory diseases.

CD and ulcerative colitis (UC) are inflammatory bowel diseases (IBD), consequential to a dysregulated mucosal inflammatory response (20). The intestinal immune compartment is differentially regulated and its antigenic repertoire is independently shaped to accommodate the heavy antigenic load characteristic of the gut environment (21). In IBD, tolerance to intestinal Ags is perturbed (22, 23) and strong evidence implicates a skewed T cell-mediated Th1 response in CD (24) as well as mouse models of IBD (25, 26). Anti-TNF therapy is used to treat Th1-mediated rheumatoid arthritis (27) and CD patients (28). However, the partial success of blocking TNF alone in only a subset of CD patients emphasized the complexity of mucosal immune regulatory mechanisms and prompted an investigation of other TNF superfamily members in human IBD pathology (20, 29).

In the present study, we characterized the potential for LIGHT expression in subsets of CD4+ T cells from patients with IBD pathology. The results reveal enhanced potential of LIGHT expression by mature Th1 CD4+ T cells and in T cells of the mucosal compartment. Furthermore, LIGHT-HVEM signaling induced IFN-{gamma} production in vitro in the absence of additional stimulation and can mediate T-T interactions augmenting CD2-mediated IFN-{gamma} synthesis by intestinal T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cohort and specimen procurement

Blood leukocytes were obtained by venipuncture from healthy adult volunteers and from intestinal specimens from patients undergoing intestinal resection for clinical reasons. Patient diagnosis was defined as CD, UC. or non-IBD using standard clinical, radiographic, and endoscopic criteria (30), and gross tissue involvement was validated microscopically. Patients treated with cyclosporin A and patients with indeterminate colitis were omitted from the study. Procedures for subject recruitment, informed consent, and specimen procurement were in accordance with protocols approved by the Institutional Review Board for Human Subject Protection of the Cedars-Sinai Medical Center.

Cell isolation and culture

PBMC were isolated from uncoagulated blood by standard Ficoll-Hypaque density gradient centrifugation. Mononuclear cells from lamina propria (LPMC) were isolated as described previously (31). Briefly, epithelial cells were removed by washing in EDTA, followed by enzymatic disruption of the lamina propria (LP) matrix, mincing, and density gradient purification of LPMC. Lymphocytes were cultured at 0.25–1 x 106 cells/ml in RPMI 1640 containing 2 mM L-glutamine and 25 mM HEPES buffer (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals, Norcross, GA), 50 µg/ml gentamicin (Omega Scientific, Tarzana, CA), and LPMC with additional 0.25 µg/ml amphotericin B (Gemini Bio-Products, Woodland, CA). Where indicated, lymphocytes were stimulated by 40 ng/ml PMA and 1 µg/ml ionomycin (Sigma-Aldrich, St. Louis, MO) or by Ab cross-linking of cell surface CD2 and/or HVEM.

T cell subset purification

Nonlymphoid cells were partially depleted from LPMC and PBMC preparations by adherence to plastic for 16–20 h before further purification or experimentation. CD4+, CD8+, CD4+CD45RA, and CD4+CD45RO T cells were purified from PBMC by flow cytometry (FACStar; BD Biosciences, Franklin Lakes, NJ) gating on CD3+ staining for the CD4/8+ subsets or on CD4+ staining for the CD45RA/RO subsets. Purity was consistently >99% for the gated markers when reanalyzed by flow cytometry (FACScan; BD Biosciences).

Ab reagents

Gem1A.1 is an anti-human LIGHT combinatorial Ab containing VH and V{kappa} chains generated from a BALB/c mouse immunized with recombinant LIGHT (32). Gem1A.1 is recognized by anti-mouse Ig {kappa}. Mouse antimethamphetamine was used as an isotype control and was provided by G. Valkirs (Biosite Diagnostics, San Diego, CA). Recombinant soluble LIGHT (LIGHTt66) was prepared as described previously (33). II-23.D7 human CD4+ T cell hybridoma is responsive to stimulation with phorbol ester and ionomycin (34); HEK293 cells stably transduced with human LIGHT cDNA have been described elsewhere (33).

A polyclonal goat anti-HVEM was generated by immunization with purified human HVEM-Fc, the serum was absorbed with immobilized human IgG, and purified IgG was prepared by protein G affinity chromatography. Anti-LIGHT Ab was used at 20 µg/ml in blocking experiments and anti-HVEM was used at 0.2 µg/ml for T cell stimulation. The anti-CD2 mAb pair (clones GD10 and CB6, used at 0.5 µg/ml final each) was a gift from Dr. C. D. Benjamin (Biogen, Cambridge, MA). Additional chromophore-conjugated Abs specific for human CD3, CD4, CD8, CD45RA, CD45RO, OX40, CD40L, CTLA4, p55, and IFN-{gamma} were obtained from Caltag Laboratories (Burlingame, CA).

Cell staining for flow cytometry

Cells were blocked with goat IgG for 20 min on ice, then indirectly stained for membrane-associated LIGHT using a recombinant mouse anti-human LIGHT Fab or isotype control Fab (Jackson ImmunoResearch, West Grove, PA) and detected after washing by a FITC-conjugated goat anti-mouse (H and L) Fab (Jackson ImmunoResearch) for 30 min per step. After washing and blocking with mouse IgG for 20 min, cells were stained for additional surface markers for 20 min on ice. Alternatively, cells were lightly fixed and permabilized for staining of intracellular IFN-{gamma} using Fix and Perm reagents and protocols (Caltag Laboratories). Flow cytometric analysis included 20,000–50,000 events on a FACScan (BD Biosciences) and CellQuest analytical software. Nonspecific staining by control isotypes was subtracted from the percentage of specific staining for each cell subset when reported numerically as mean fluorescence change.

IFN-{gamma} ELISA

IFN-{gamma} was quantified in culture supernatants by amplified sandwich ELISA as previously reported (31), and concentration was calculated relative to a standard (recombinant human IFN-{gamma}; R&D Systems, Minneapolis, MN). Briefly, secreted IFN-{gamma} was captured by a plate coated with anti-IFN-{gamma} mAb (BD PharMingen, San Diego, CA) and detected by binding of a second anti-IFN-{gamma}-biotinylated mAb (BD PharMingen), followed by streptavidin-alkaline phosphatase (Jackson ImmunoResearch) and 0.2 mM NADPH substrate (Sigma-Aldrich). The signal was amplified using 3% 2-propanol, 1 mM iodonitrotetrazoliun violet, 75 µg/ml alcohol dehydrogenase, and 50 µg/ml diaphorase (Sigma-Aldrich) and was measured at 490 nm by an Emax plate reader (Molecular Devices, Menlo Park, CA) and ELISA Master vintage software (R. L. Deem, Cedars-Sinai Medical Center, Los Angeles, CA).

Real-time RT-PCR

Cells were lysed in guanidium thiocyanate buffer and total RNA was isolated using RNeasy kits (Qiagen, Valencia, CA). For micropurification of RNA from <105 cells, lysates were supplemented with 12 µg of rRNA as a carrier (Sigma-Aldrich). LIGHT mRNA levels were quantified by real-time RT-PCR (iCycler; Bio-Rad, Hercules, CA) using One-Step RT-PCR mixture (Qiagen) with a LIGHT-specific dual-labeled probe and intron spanning primers normalized to a primer limiting 18S ribosomal RNA amplification measured in duplex. The following primer (Integrated DNA Technologies, Coralville, IA) and probe (Operon, Valencia, CA) sets were designed using Primer 3 software (35): LIGHT forward primer, 5'-TGGCGTCTAGGAGAGATGGT-3'; reverse primer, 5'-GGTTGACCTCGTGAGACCTT-3'; Hyb probe, 5'-6-FAM-AGCTGCTCCCAGGAGCCTGC-BHQ1–3'; 18S forward primer, 5'-AAACGGCTACCACATCCAAG-3'; reverse primer, 5'-CCTCCAATGGATCCTCGTTA-3'; and Hyb probe, 5'-TxRed-AGCAGGCGCGCAAATTACCC-BHQ3–3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of LIGHT-expressing T cell subsets in peripheral blood (PB)

Mouse models indicate that T cell expression of LIGHT plays a pathological role in inflammatory processes, suggesting that LIGHT may be expressed by specific T cell subsets. T cells from human PB T cells were examined for expression of LIGHT following 24 h of stimulation with PMA and ionomycin (P/I). Cell surface LIGHT was detected by staining with a combinatorial mouse anti-human LIGHT Fab (GemA1.1) (10) and flow cytometry. This reagent specifically stained HEK293 cells stably transfected with LIGHT cDNA (Fig. 1), but not untransfected HEK293 cells (data not shown). The expression of LIGHT by II-23 T cells requires stimulation with both P/I (34) and as predicted GemA1.1-stained P/I activated II-23 T cell hybridoma cells, but not unactivated cells (Fig. 1). These results demonstrated that the anti-LIGHT recognizes recombinant and membrane-bound endogenous LIGHT.



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  		FIGURE 1. Gem1A.1 binds human LIGHT. HEK293 cells stably expressing transfected LIGHT (left panel), unactivated (center panel), or P/I-activated II-23 T cells (4 h; right panel) were incubated with 5 µg/ml Gem1A.1 (solid line) or isotype control (dashed line), and binding was detected with goat anti-mouse {kappa} chain-PE and analyzed by flow cytometry.

 
LIGHT was prominently up-regulated on CD8+ T cells and on a subset of CD4+ T cells derived from PB (Fig. 2A), although less intense staining was observed on CD4+ cells. Previous work indicated both T cell populations when activated with P/I bind HVEM-Fc, indicating LIGHT is in a receptor competent form (34). Levels of LIGHT expression by highly purified P/I-activated CD4+ or CD8+ T cells was similar to unfractionated PBL, thus excluding secondary cell types as contributors to LIGHT expression by T cells (Fig. 2B). Flow cytometric detection of LIGHT was corroborated by analysis of LIGHT mRNA levels in purified T cell populations by RT-PCR. LIGHT mRNA was not detectable in resting T cell preparations, but was readily induced by P/I to peak levels within 30–60 min and stable for >24 h (Fig. 2C). LIGHT mRNA levels in purified CD4+ as well as CD8+ or unseparated CD3+ T cells were again similar to unseparated PB following stimulation with P/I. These findings indicate that LIGHT expression is regulated at the transcriptional level in T cells.



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  		FIGURE 2. LIGHT is induced at the protein and mRNA level on activated CD4+ and CD8+ T cells. A, PBMC were activated in vitro with P/I for 16 h and surface stained with anti-LIGHT Ab (shaded) or isotype control Ab (unshaded), anti-CD3 and anti-CD4/8. Gated CD3+CD4+ or CD3+CD8+ histograms are shown. B, CD3+CD4+ or CD3+CD8+ were purified by flow cytometry (>99%) before activation and stained with anti-LIGHT Ab or isotype control. C, Unseparated PBMC and flow-sorted CD3+, CD3+CD4+, and CD3+CD8+ were activated with P/I for 16 h and LIGHT mRNA levels were quantified by real-time RT-PCR in duplex with primer-limiting 18S rRNA amplification. 18S adjusted threshold cycle for LIGHT signal and a 50-cycle end point agarose gel is presented. Data are representative of a minimum of three experiments. ND, Not detected.

 
Flow cytometric analysis of LIGHT on activated T cells indicated a wide range of expression levels for individual CD4+ T cells (Fig. 2). We, therefore, tested whether LIGHT expression was associated with known CD4+ subsets. The expression of CD45RO is an indicator of previous Ag exposure and mature/effector CD4+ T cell phenotype, whereas CD45RA defines a naive population (36). LIGHT expression was significantly higher (and almost exclusive) in the CD4+/CD45RO subset when compared with the CD4+/CD45RA cell subset (Fig. 3A). Moreover, LIGHT expression on CD4+/CD45RO T cells was as high as CD8+ T cells (Figs. 2 and 4). These observations indicate that a subset of CD4+ T cells is able to attain a high level of LIGHT expression previously believed exclusive to CD8+ T cells (37). Another study (38) indicated that non-naive T cells expressed intracellular LIGHT but did not distinguish which isoform of LIGHT was detected (10).



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  		FIGURE 3. LIGHT is induced at the protein and mRNA level on mature CD4+/CD45RO but not on naive CD4+/CD45RA T cells. A, PBMC were activated in vitro with P/I for 16 h and surface stained with anti-LIGHT Ab (shaded) or isotype control Ab (unshaded), anti-CD4 and anti-CD45RO, or anti-CD45RA. Lymphocyte-gated CD4+ histograms are shown. B, Unseparated PBMC and flow-sorted CD3+CD4+CD45RO or CD3+CD4+CD45RA (>99% purity) were activated with P/I for 16 h and LIGHT mRNA levels were quantified by real-time RT-PCR in duplex with primer-limiting 18S rRNA amplification. 18S adjusted threshold cycle for LIGHT signal and a 50-cycle end point agarose gel is presented. Data are representative of a minimum of three experiments. ND, Not detected.

 


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  		FIGURE 4. LIGHT protein up-regulation partially correlates with activation markers OX40 and CD40L on CD4+ but not CD8+ T cells. PBMC were activated in vitro with P/I for 16 h and surface stained with anti-LIGHT Ab (shaded) or isotype control Ab (unshaded), anti-CD4/8 and anti-OX40, or anti-CD40L. Lymphocyte-gated CD4+ or CD8+ histograms are shown for A (OX40) and B (CD40L).

 
Up-regulation of the transmembrane isoform of LIGHT was validated at the mRNA level by quantitative real-time RT-PCR analysis using a specific primer probe set in highly purified (flow sorted to >99% purity) CD45RO or CD45RA preparations of CD4+ T cells. Marginal levels of LIGHT mRNA were detected in activated CD4+ CD45RA T cells, while significantly higher levels were detected in the activated CD45RO subset (~8- to 10-fold more LIGHT mRNA per cell), directly correlating with membrane LIGHT protein expression in these cell subsets (Fig. 3B). Interestingly, LIGHT mRNA encoding the transmembrane domain was never detected in resting T cells (Figs. 2C and 3B), suggesting that intracellular LIGHT protein reported in those cells (37, 38) could be an isotype lacking that domain, although variant LIGHT mRNA was not consistently detected in our system (10).

Mature CD4+/CD45RO T cells are more responsive to activating stimuli, and LIGHT expression in this subset could reflect enhanced responsiveness rather than the state of differentiation. In accord, we tested for LIGHT correlation with T cell activation as marked by up-regulation of surface markers OX40 (CD134) and CD40L (CD154) (39, 40). PBL were surface stained for CD4/8, LIGHT, and OX40 or CD40L following P/I activation, indicating higher LIGHT expression levels in CD4+ T cells expressing OX40 (Fig. 4A) or CD40L (Fig. 4B). However, LIGHT up-regulation only partially tracked with cells expressing OX40 or CD40L, and significant LIGHT expression was detected in cells lacking the OX40 and CD40L, thus suggesting that LIGHT expression is governed by factors independent of individual cell responsiveness.

LIGHT-HVEM signals can enhance IFN-{gamma} production by T cells (41) and could be a key mediator of T-T interaction driving a proinflammatory Th1 response (36). We, therefore, examined whether LIGHT is preferentially expressed by Th1 CD4+ T cells defined by IFN-{gamma} expression. IFN-{gamma} production was analyzed by intracellular staining following activation by P/I in the presence of brefeldin A. Multiparameter analysis of LIGHT expression in the CD3, CD4, or CD8 T cell subsets indicated LIGHT was primarily coexpressed by IFN-{gamma}+ T cells, with an almost exclusive expression of LIGHT in the IFN-{gamma}-producing subset of CD4+ T cells (Fig. 5A). Consistent LIGHT-IFN-{gamma} coexpression in pure T cell preparations (>99%) precluded a contribution from a secondary cell type as an underlying mechanism (Fig. 5B).



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  		FIGURE 5. Membrane LIGHT protein is induced primarily on IFN-{gamma}-producing T cells in the absence of accessory cells. A, PBMC were activated in vitro with P/I for 16 h and surface stained with anti-LIGHT Ab (shaded) or isotype control Ab (unshaded), anti-CD4/8, and intracellularly stained with anti-IFN-{gamma}. Gated CD3+CD4+ or CD3+CD8+ histograms are shown for the IFN-{gamma}+ and IFN-{gamma} subsets. B, CD3+CD4+ or CD3+CD8+ were purified by flow cytometry (>99%) before activation and stained with anti-LIGHT Ab or isotype control and intracellular anti-IFN-{gamma}.

 
LIGHT exhibits a unique expression profile in the mucosal compartment

The human intestinal mucosa may be a primary site for LIGHT-mediated proinflammatory activity. The expression profile of LIGHT was examined on T cells derived from the human intestinal immune compartment including the mesenteric lymph nodes or the LP of the rectum, colon, and the small bowel. The time course of LIGHT induction following P/I activation of CD4+ or CD8+ T cells from these tissues revealed a rapid induction of LIGHT on LP T cells, reaching maximal cell surface levels by 6 h on cells from the colon or 1–2 h in the rectum or small bowel (Fig. 6, C and D). By contrast, LIGHT induction on donor-matched T cells from PB or mesenteric lymph nodes was slower and linear for up to 24 h (Fig. 6, A and B) (37). LIGHT induction was confirmed at the mRNA level, although the time course of induction did not differ significantly between intestinal LP lymphocytes (LPL) and lymph nodes or PBL preparations, all peaking by 1 h (data not shown), suggesting that posttranscriptional mechanisms can control LIGHT expression.



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  		FIGURE 6. Intestinal LP T cell induce LIGHT earlier than T cells from the PB or mesenteric lymph nodes. Lymphocytes were isolated as described in Materials and Methods and activated with P/I for the indicated time. Cells were then surface stained with anti-LIGHT Ab or isotype control Ab and anti-CD3/8. CD4+ T cells were defined as CD3+CD8– and mean fluorescence changes from the isotype control are plotted for CD4+ and CD8+ T cells from A, PB; B, mesenteric lymph node; C, colonic LP; and D, small bowel LP.

 
Interestingly, although rapid induction of LIGHT was shared by LP T cells from all intestinal sites tested, protein levels were only induced to significantly higher peak levels in LP CD4+ and CD8+ T cells from the small bowel or the rectum, but not the colon when compared with lymph node or PB T cells (Fig. 7A). Most striking was the high peak levels of LIGHT in the CD4+ subset from the small bowel or rectum, reaching levels as high as typically seen in PB CD8+ or CD4+/CD45RO T cells (Figs. 2, 3, and 7).



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  		FIGURE 7. Maximal membrane LIGHT fluorescence intensity is higher in small bowel and rectal LP T cells. Lymphocytes were isolated as described in Materials and Methods and activated with P/I for 24 h (PBL) or 6 h (LPL). Cells were then surface stained with anti-LIGHT Ab or isotype control Ab and anti-CD3/8. CD4+ T cells were defined as CD3+CD8– and mean fluorescence changes from the isotype control are plotted for CD4+ and CD8+ T cells. A, Comparison of membrane LIGHT expression between CD4+ and CD8+ T cells of the PB, mesenteric lymph node (LN), and the LP of the rectum (Rec), colon, and small bowel (SB). Student two-sided t test p values are tabulated when statistically significant. B, Analysis of membrane LIGHT expression in CD4+ and CD8+ T cells from inflamed vs uninvolved, mesenteric lymph node or the LP of the rectum, colon, and small bowel. C, Analysis of membrane LIGHT expression in CD4+ and CD8+ T cells from CD, UC, or non-IBD disease groups.

 
The relatively rapid time course of LIGHT induction by mucosal T cells suggested that increased expression may lead to enhanced signaling activity in the gut and thus serve as a correlate for disease activity. Accordingly, we examined whether LIGHT expression levels correlated with differences in T cell activation characteristics between gut location and disease state. LIGHT expression on in vitro-activated T cells was analyzed by surface staining and flow cytometry in a cohort of patients with IBD stratified according to the clinical status (Table I). However, LP T cells from grossly inflamed mucosal sites expressed only slightly higher LIGHT when compared with uninvolved sites, with mean fluorescence intensity differences not statistically significant for this sample size (Fig. 7B). Additional comparison of peak levels of LIGHT in LP T cells from CD, UC and non-IBD patients did not segregate these disease groups (Fig. 7C). We thus conclude that differential LIGHT regulation by intestinal T cells is plausible given their distinctively rapid induction profile and higher peak levels, although the in vitro-induced LIGHT peak levels may not reflect disease state or tissue involvement.


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  		Table I. IBD intestinal specimen cohorta

 
LIGHT-HVEM regulates IFN-{gamma} production as a component of CD2-mediated activation

CD2 responsiveness has been proposed as a unique character of LP T cells (42), providing a gut-specific mechanism of IFN-{gamma} activation (43). We investigated whether LIGHT can contribute to intestinal inflammation by augmenting LP T cell production of IFN-{gamma} in the context of CD2-mediated activation. Isolated human LPL were activated in vitro by anti-CD2 cross-linking or P/I in the presence of a blocking anti-LIGHT or isotype control Abs, and secreted IFN-{gamma} was measured in culture supernatants by ELISA (Fig. 8A). Activation with anti-CD2 stimulation induced modest amounts of IFN-{gamma}; however, those levels were significantly reduced in the presence of LIGHT-blocking Abs, although variation was seen between donors (0–45% inhibition for 12 intestinal samples tested). Costimulation by anti-CD28 Abs induced maximal IFN-{gamma} production and abrogated anti-LIGHT inhibition of CD2-mediated IFN-{gamma} synthesis (Fig. 8A).



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  		FIGURE 8. Signal via the LIGHT receptor, HVEM independently induces IFN-{gamma} and partakes in CD2-mediated IFN-{gamma} production by LP T cells. LP T cells were activated for 24 h. Secreted IFN-{gamma} in culture supernatants was measured by sandwich ELISA and quantified based on a standard curve. A, LPL were activated with anti-CD2 Abs, anti-CD2 and anti-CD28, or P/I in the presence of anti-LIGHT Ab (GemA.1.1; 20 µg/ml) or isotype control Abs. B, Inflamed, uninvolved, and non-IBD LPL were activated with an anti-CD2 Ab pair or an agonistic anti-HVEM Ab in solution. C, LPL were activated with an anti-CD2 Ab or anti-CD2 and anti-CD28 in the presence of an agonistic anti-HVEM Ab or isotype control Abs. Means and SDs for three representative experiments are plotted.

 
Interestingly, IFN-{gamma} production was induced by cultured LPL stimulated with recombinant soluble human LIGHT (LIGHTt66) or an agonistic anti-HVEM Ab in the absence of TCR/CD3 or CD2 signal (Fig. 8B). Furthermore, stimulation by LIGHTt66 or anti-HVEM did not further augment CD2-mediated induction of IFN-{gamma} and showed no synergy with CD28-mediated costimulation (Fig. 8C). Finally, since CD2 mediates Ag-independent T cell activation in the intestine, LIGHT could play a role in autoimmune pathology by inducing Ag-independent T cell activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The intestinal immune compartment is tightly regulated to prevent excessive reactivity to the heavy antigenic load characteristic of the gut environment (20, 23, 44). Perturbation in these mucosal immunoregulatory mechanisms may lead to breakdown of tolerance to intestinal Ags and an ensuing chronic mucosal inflammation (22, 23, 45). Several studies established mucosal CD4+ T cells and, in particular, those with specificity to bacterial Ags (46), as key mediators of the aberrant inflammatory response underlying IBD. More specifically, CD4+ CD45RBhigh, Th1, and a deficiency in T regulatory type 1 cells have been implicated in IBD pathology (18, 24, 25, 47). However, the characteristics of these pathogenic T cells and mechanism by which breakdown of tolerance occurs remain largely unknown. Signaling via TNF family members is a key costimulatory mechanism modulating T cell responses to a TCR and MHC peptide engagement, which is critical during T cell maturation, and several specific TNF family members have been implicated in thymic T cell selection (1, 3). Of those, LIGHT recently emerged as a significant mediator of T cell development and negative selection (15), which also plays a unique regulatory role within the mucosal immune compartment (6, 7). Consequently, since the repertoire of the mucosal immune compartment is continuously and independently reconstructed (21), LIGHT-mediated signaling could play a role in mucosal T cell selection and thus partake in the shaping of pathological immune repertoire underlying IBD (22). Our observations described here demonstrate that mucosal T cells express LIGHT rapidly after cellular activation and that the CD45RO memory/effector population is selectively differentiated to express LIGHT. Moreover, the requirement of LIGHT for IFN-{gamma} production and the direct ability of HVEM to induce IFN-{gamma} support the notion that LIGHT is a putative mediator of proinflammatory T-T interactions in the intestinal mucosa.

LIGHT expression levels as detected by flow cytometry in the activated CD4+ T cell population was heterogeneous, in contrast to the more uniform level on activated CD8+ T cells (Fig. 2). This heterogeneity arises because a relatively small subset of CD4+ T cells up-regulate LIGHT to levels similar to those of CD8+ cells, while the majority expresses little or no LIGHT at all. We demonstrated that LIGHT expression is restricted to the mature CD4+ CD45RO subset, a population with previous antigenic exposure and thus highly pertinent to IBD pathology (Fig. 3). Partial correlation between LIGHT expression levels and expression of T cell activation markers indicated that variation in LIGHT expression levels may reflect activation state to a degree, but that additional factors contribute to the potential up-regulation of cell surface LIGHT (Fig. 4).

Intensive immune interface with intestinal Ags plays a key role in IBD pathology (23, 46, 48). However, T cell maturation and repertoire shaping via Ag exposure is not sufficient in itself for the induction of an inflammatory response, given that Ag-specific T cells can be regulatory, as well as effector cells (49). Thus, a proper balance between tolerogenic and nontolerogenic specificities is essential to immune homeostasis and protective vs pathogenic responses. In CD, this balance is lost and data suggest an unopposed Th1 response as an underlying mechanism (24, 25). Our findings here indicate almost exclusive LIGHT expression by IFN-{gamma}-producing CD4+ T cells and suggest a role for LIGHT in mediating a Th1 response, thus further tying LIGHT with CD pathology (Fig. 5). LIGHT signaling via HVEM and the LT{beta}R activates major signaling cascades and modulates transcription via NF-{kappa}B as well as NF-{kappa}B-independent mechanisms. For example, Morel et al. (37) described LIGHT-mediated down-regulation of HVEM, and Lee et al. (50) reported LIGHT-mediated induction of TNF and IL-8 in the THP1 monocytic cell line model. Morel et al. (38) recently reported a role for T cell-derived LIGHT in the production of IL-12 by activated dendritic cells. In T cells, LIGHT has been reported to provide costimulation augmenting proliferation and cytokine production (14), and our data demonstrate for the first time that HVEM signaling by itself induces IFN-{gamma} production in LP T cells (Fig. 8B). Consequently, LIGHT expression by IFN-{gamma}-producing T cells may initiate a self-propagating loop, escalating a Th1 response via T-T interaction. Furthermore, LIGHT can be induced on LP NK cells (mean fluorescence intensity, 15–20), which produce abundant amounts of IFN-{gamma} and play an important immunoregulatory role (51).

HVEM is constitutively expressed on most LP T cells, consistent with previous reports in PB T cells (14). The specific mechanisms regulating LIGHT expression are yet to be elucidated, but the correlation of LIGHT expression with IFN-{gamma} production may point to a shared regulatory pathway. In this study, we report more rapid kinetics of LIGHT up-regulation and higher peak levels on LP T cells than PB T cells (Fig. 6). Interestingly, IFN-{gamma} is also differentially regulated at the transcriptional level in the intestinal T cell compartment (43). Considering the significance of LIGHT-mediated signaling to IFN-{gamma} production (52), it is plausible that sustained or elevated LIGHT expression in the intestine could contribute to the gut-specific regulation of IFN-{gamma}.

Rapid kinetics of LIGHT up-regulation on T cells as well as higher peak levels (Figs. 6 and 7) may account for an increased activity of LIGHT within the intestinal immune compartment. In addition, higher peak levels of cell surface LIGHT were recorded in small bowel T cells when compared with colonic or peripheral T cells. Interestingly, these observations further support a linkage between LIGHT and IFN-{gamma} pathways since the small bowel is the primary affected organ in Th1-mediated CD, while LIGHT peak levels were lower in the colon, which is the primary target in UC for which there is less evidence of a Th1-mediated pathology (24). In addition, elevated LIGHT expression in mucosal T cells agree with our data localizing LIGHT expression to mature T cells considering the massive antigenic exposure unique to the gut immune compartment. The CD4+ CD45RO subset of T cells is more prominent in the mucosal compartment (RO/RA 2:1 in the LP vs 1:2 in the periphery) (31), and since these are the LIGHT-expressing cells, their increased abundance may account for the unique expression profile of LIGHT in LP T cells.

Constitutive T cell expression of LIGHT in the mouse resulted in mucosal inflammation and provided the initial evidence for LIGHT role in mucosal immune regulation (6, 7). Elevated LIGHT expression on human intestinal T cells further supports a proinflammatory role for LIGHT in human IBD pathology (Fig. 7). In this study, LIGHT expression levels following maximal in vitro activation of T cells from IBD mucosa was similar to controls. Nonetheless, cell surface protein expression is abolished during enzymatic disruption of the intestinal mucosa and activation-induced expression in vitro may not directly reflect in vivo expression. Thus, LIGHT proinflammatory signaling or function in T cell selection may still play a significant role propagating a primary pathological event such as elevated antigenic sampling, leading to a pathological immune repertoire and an aberrant inflammatory response.

In summary, our findings demonstrate for the first time that LIGHT can be expressed at high levels on human mucosal CD4+ T cells. Furthermore, LIGHT expression was localized to the mature Th1-type CD4+ T cells, key cellular effectors of an inflammatory response and crucial to IBD pathogenesis (24, 25, 26). In addition, we identified a more rapid induction and higher peak levels of LIGHT on human mucosal T cells, which is especially interesting since data from transgenic mouse studies indicated LIGHT-dependent inflammation selectively targeted the intestine. In conclusion, the mucosal specificity of LIGHT-mediated inflammation could have significant pathological implications in human IBD, and thus merits further investigation of gut-specific immune regulatory mechanisms.


   Acknowledgments
 
We acknowledge Walker R. Force (Gemini Sciences) for assistance with the anti-human LIGHT Ab. We thank John L. Prehn for helpful discussions and critical reading of this manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants F32DK10139 (to O.C.), R01DK57328/R01DK43211 (to S.R.T.), and R37AI33068 (to C.F.W.). Back

2 Address correspondence and reprint requests to Dr. Stephan R. Targan, Cedars-Sinai Inflammatory Bowel Disease Center, 8700 Beverly Boulevard, Suite D4063, Los Angeles, CA 90048. E-mail address: targans{at}cshs.org Back

3 Abbreviations used in this paper: LIGHT, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; LP, lamina propria; LPL, LP lymphocyte; P/I, PMA and ionomycin; LT, lymphotoxin; HVEM, herpesvirus entry mediator; LPMC LP mononuclear cell; PB, peripheral blood. Back

Received for publication January 9, 2004. Accepted for publication April 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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