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J Biol Chem, Vol. 275, Issue 19, 14307-14315, May 12, 2000

The Lymphotoxin- Receptor Is Necessary and Sufficient for LIGHT-mediated Apoptosis of Tumor Cells^*

Isabelle A. Rooney, Kris D. Butrovich, Alison A. Glass, Stephen Borboroglu, Chris A. Benedict, J. Charles Whitbeck^§, Gary H. Cohen^§, Roselyn J. Eisenberg^§, and Carl F. Ware^¶

From the  Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121 and the ^§ Department of Microbiology and Center for Oral Health Research, School of Dental Medicine and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin- receptor (LTR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A'  A" and D  E loops of LIGHT, which altered binding to LTR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTR interaction and implicates a distinct biological role for LIGHT-HveA system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytokines related to tumor necrosis factor (TNF)¹ mediate developmental and effector functions of the innate and adaptive immune systems. Signaling by TNF-related cytokines is initiated by aggregation of specific cell surface receptors. TNF, lymphotoxin  (LT), and LT and the recently identified protein LIGHT exhibit distinct but overlapping patterns of binding to four cognate cell surface receptors that together define a core group within the larger TNF superfamily. TNF and LT are homotrimeric ligands that bind two receptors, TNFR1 (55-60 kDa; CD120a) and TNFR2 (75-80 kDa; CD120b) (1). LT also forms heterotrimers with LT (2), where the predominant form expressed by activated T cells is LT₁₂, which specifically binds the LTR (3, 4). LIGHT engages the herpesvirus entry mediator, HveA (also known as HVEM) (5, 6). The shared receptor binding patterns among these cytokines are observed with LIGHT binding LTR and HveA binding LT but not TNF or LT heterotrimer. Although the complexity of receptor cross-utilization suggests functional redundancy of these cytokines, gene deletion studies in mice have revealed unique and cooperating roles for the LT and TNF ligand-receptor systems in the development and function of the immune system.

The LT-LTR system is required for the formation of lymphoid tissue (lymph nodes and Peyer's patches) as well as the segregation of T and B-lymphocytes into distinct compartments in the spleen and the formation of germinal centers (7). Lymphoid tissue is largely unaffected by deletion of TNF, TNFR1, or TNFR2; however, TNF and TNFR1 are important for correct formation of germinal centers (8-10). Roles for LIGHT and HveA have not yet been revealed by gene deletion studies; however, phenotypic differences between LT- and LT-deficient mice (11, 12) as well as LT- and LTR-deficient mice (13) implicate LIGHT/HveA signaling in some aspects of lymphoid tissue organization.

That LIGHT engages both HveA and LTR raises the question of whether these receptors signal independently or cooperatively. LTR stimulates expression of adhesion molecules (14, 15) and induces apoptosis in adenocarcinoma cell lines when bound by LT₁₂ (16). Unlike the death domain-containing TNFRs (e.g. Fas (17, 18)), which initiate direct activation of the caspases leading to rapid apoptosis, the LTR induces a slow apoptotic death (16), similar to TNFR2 (19) and CD30 (20, 21). Recent evidence suggests that apoptosis mediated by CD40 and TNFR2 occurs indirectly through the induction of TNF and activation of the TNFR1 pathway (22).

LTR and HveA signal via TRAF molecules, a family of six RING finger proteins that bind directly to cytosolic domains of these receptors, allowing the propagation of signals to downstream effectors (23, 24). For example, TRAF2 and TRAF5 act as adapters for NIK, a kinase that activates the IB kinases generating the transcriptionally active form of NF-B (25-27). TRAF3 is involved in the propagation of signals via the LTR that activate cell death (28-30). HveA also binds TRAF2 and TRAF5, which do not induce apoptosis, but activate NF-B and JNK/AP1 pathways (31, 32). LIGHT induces growth arrest of HT29 cells (33) and, as a transfected cDNA, inhibits growth of some tumors in mice (34), and it may serve a costimulatory role in lymphocyte activation (33). However, the relative contribution of HveA and LTR to these LIGHT-mediated effects has not been established.

Here, we have generated mutants of LIGHT that discriminate between its two receptors and utilized receptor-specific antibodies to reveal an indispensable role of the LTR, but not HveA, in the apoptotic and integrin-inducing effects of LIGHT.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Reagents-- The HT29.14S cell line is a clone of the HT29 colon adenocarcinoma sensitive to the proapoptotic activity of TNF-related ligands (16). HT29.14S cells transduced with retroviral vectors that express the TRAF3 dominant negative mutant T31-339 or empty vector as a control were generated as previously described (29). Human embryonic kidney (HEK293) cells and 293T cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM containing 10% fetal bovine serum with glutamine and penicillin/streptomycin. Normal human dermal fibroblasts (NHDF) from neonatal foreskins were purchased from Clonetics (San Diego, CA) and grown in DMEM supplemented with 10% fetal bovine serum, insulin (5 µg/ml), and fibroblast growth factor (1 ng/ml) (Sigma).

Recombinant human interferon- (IFN-) and TNF were gifts of Dr. J. Browning (Biogen, Inc., Cambridge, MA). Fusion proteins constructed with the Fc region of human IgG₁ and human HveA (HveA-Fc) (6), LTR (LTR-Fc) (3), TNF-R1-Fc (35, 36), and TRAIL R1-Fc (37) (gift from J. Tschopp) were prepared as described previously. Human LT₁₂ was expressed in insect cells as described by Browning et al. (38) and purified by ion exchange on a SP Hitrap column (Amersham Pharmacia Biotech) followed by affinity purification with LTR-Fc coupled to Affi-Gel (Bio-Rad) and depletion of contaminating ligands (mainly LT21) by TNFR1-Fc affinity matrix (39).

Antibodies to HveA or LTR (28) were produced by immunizing goats with HveA-Fc or LTR-Fc fusion proteins as described previously (40). Mouse monoclonal anti-LTR antibodies BDA8 (IgG1) and CDH10 (IgG1) were gifts of Dr. J. Browning (Biogen, Inc.) (41). FLAG epitope-specific mAb M2 (anti-FLAG) was purchased from Sigma. Anti-ICAM-1 (P2A4) mAb was obtained from Chemicon International, Inc. Mouse monoclonal anti-HveA antibodies CW1 and CW8 were produced in mice with purified ectodomain of HveA. Details concerning the properties of these antibodies will be presented elsewhere.² Sheep anti-mouse IgG coupled to horseradish peroxidase was purchased from Amersham Pharmacia Biotech.

Production of a Soluble Form of LIGHT (LIGHTt66)-- Full-length LIGHT was cloned from activated II23.D7 T cell hybridoma cells by reverse transcriptase-PCR using the following primer: forward, 5'-TATAAGCTTGAGGTTGAAGGACCCAGG-3'; reverse, 5'-CAGGGATCCCTTCCTTCACACCATGAAAGC-3' (6). The LIGHT PCR product was subcloned into pCDNA3.1(+) to create pCDNA3.1-LIGHT. The extracellular domain (encoding Gly⁶⁶ to Val²⁴⁰) was amplified from pCDNA3-LIGHT by PCR using the following primers: forward, 5'-GTAGGAGAGATGGTCACCCGCCT-3'; reverse, 5'-GGAACGCGAATTCCCACGTGTCAGACCCATGTCCAAT-3'. The amplified LIGHT product was digested with EcoRI and ligated into the SnaB1 and EcoRI sites of pCDNA3.1-VCAM-FLAG, which contains the VCAM1 signal sequence fused to the FLAG epitope, 5' of the FLAG epitope.

HEK293 cells were transfected by the calcium phosphate method, and stable clones were selected with G418 and screened for LIGHT production. LIGHTt66 was purified from culture supernatants of cells grown in DMEM containing 0.5% fetal bovine serum. LIGHTt66 was purified by ion exchange chromatography with an SP Hitrap column (Amersham Pharmacia Biotech) and affinity chromatography with anti-FLAG (M2) coupled to Affi-Gel (Bio-Rad). LIGHTt66 was eluted from the column using 20 mM glycine, 150 mM NaCl, pH 3.0, and pH-neutralized immediately by collection into 50 mM Tris, pH 7.4. Protein concentration was determined by amino acid analysis and absorbency at 280 nm.

Point Mutants of LIGHTt66-- Primer-introduced sequence modification was used to generate soluble LIGHT with the following single amino acid substitutions: G119E, L120Q, Q117T, and Y173F. Briefly, internal primers were designed to introduce a restriction site at the mutation location. Forward and reverse primers containing the mutations were used in separate PCRs to amplify two regions of soluble LIGHT. Primers were as follows: Q117T, 5'-ACGCTGGGCCTGGCCTTCCTGA-3" and 5'-ACTCTCCCATAACAGCGGCC-3'; G119E, 5'-GAGCTGGCCTTGCTGAGGGGCCT-3" and 5'-CAGCTGAGTCTCCCATAACA-3'; L120Q, 5'-CAGGCCTTCCTGAGGGGCCTCA-3' and 5'-GCCCAGCTGAGTCTCCCATAA-3'; Y173F, 5'-TTCCCCGAGGAGCTGGAGCT-3' and 5'-GCGGGGTGTGCGCTTGTAGA-3'.

The PCR products were ligated at the primer-introduced restriction enzyme site to create soluble LIGHT starting at amino acid Gly⁶⁶ and containing one of the 4-amino acid substitutions. The LIGHTt66 mutants were excised and ligated into VCAM-FLAG-pCDNA3.1. The VCAM FLAG-LIGHT mutant inserts were cloned into pCDNA3.1(+) (Invitrogen). All constructs were sequenced (ABI310 automated sequencer) for unambiguous verification of the mutation. LIGHTt66 mutants were produced by calcium phosphate transient transfection of 293T cells. Mutant proteins were purified from 100 ml of culture supernatant in a one-step immunoaffinity procedure using an affinity matrix of anti-FLAG antibody (M2). Protein-containing fractions were dialyzed against PBS and sterilized after dilution in medium.

ELISA for LIGHTt66-- Soluble LIGHT was measured using a capture ELISA method. The capture molecule (HveA-Fc or LTR-Fc) was coated on wells of a microtiter plate (150 ng/well in 50 µl of 150 mM NaCl, 20 mM Tris, pH 9.6) at 4 °C. After washing with PBS, 0.5% Tween 20, purified ligands diluted in PBS, 3% BSA were added to the wells and incubated for 1 h at room temperature. After washing, mouse mAb anti-FLAG (M2) (10 µg/ml in PBS/BSA) was added for 1 h at room temperature, washed, and incubated with goat anti-mouse horseradish peroxidase (1:1500) for 1 h at room temperature. Color was developed with 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma), and the OD was measured at 415 nM in a SpectraMax plate reader (Molecular Devices, Inc., Sunnyvale, CA).

Biochemical Analysis of LIGHTt66-- LIGHT was cross-linked by the addition of bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) (Pierce) at final concentrations of 5 mM or by the addition of glutaraldehyde (0.1%) for 30 min at 4 °C, and the reaction was stopped by the addition of Tris (20 mM, pH 8.0). After SDS-PAGE, proteins were transferred to polyvinylidene fluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Blots were incubated in PBS, 0.1% Tween containing 10% milk protein, 0.2% sodium azide) for 30 min. The blot was incubated with anti-FLAG M2 (5 µg/ml) and washed, and then horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse IgG; Amersham Pharmacia Biotech; 1:1000) was added for 1 h. The blot was developed using chemiluminescent detection reagents (Supersignal; Pierce). The molecular weight of native LIGHTt66 was analyzed by gel filtration on a Superose 12 column using a FPLC 500 system (Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min with collection of 0.5-ml fractions. LIGHT was detected by ELISA and by Western blot with anti-FLAG. The elution volumes of calibration proteins (blue dextran (2000 kDa), apoferitin (443 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa) were measured by absorbency at 280 nm.

Cytotoxicity Assay-- HT29.14s cells (5000/well) in DMEM were incubated at 37 °C with serial dilutions of LIGHTt66 and other cytokines in a total volume of 100 µl of DMEM in the presence or absence of 80 units/ml of human IFN-. After 72 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added, and the plate was incubated for 4 h at 37 °C. The medium was then aspirated, and 100 µl of acidified 70% isopropyl alcohol was added to dissolve the formazan product. The A₅₇₀ was measured in multiwell spectrophotometer (Spectra Max 250, Molecular Devices, Inc.). The data represent the mean ± S.D. of triplicate wells.

Molecular Modeling-- LIGHTt66 was modeled using the SwissModel server, version 3.0 (available on the World Wide Web) using 12TUN.pdb and 1TUN.pdb as the templates (42) and visualized using Rasmol (version 2.6).

Surface Plasmon Resonance-- Association and dissociation rates of the interaction of LIGHTt66 and mutants with human HveA-Fc and LTR-Fc were determined by surface plasmon resonance using a BIA-core X (BIA-core Inc., Piscataway, NJ). The capture molecule (50 µg/ml) was coupled to a CM5 sensor chip by amine coupling at pH 5.0. The sensor surface was equilibrated with PBS (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), and sensorgrams were collected at 25 °C and a flow rate of 5 µl/min. A 10-µl injection of LIGHTt66 or mutant proteins were passed over the sensor surface, and after the association phase, 800 s of dissociation data were collected. The sensor surface was regenerated after each cycle with a 10-µl pulse of 10 mM glycine pH 2.0. Sets of five analyte concentrations, 100-500 nM, were collected and analyzed by nonlinear regression using the BIAevaluation software (version 2.1). Association and dissociation data were fitted on the basis of the simple AB  A + B model.

Flow Cytometry-- HT29.14s cells or NHDF (DMEM, 3% BSA) were incubated with mouse monoclonal antibodies in a total volume of 50 µl for 30 min at 4 °C. Cells were stained with goat anti-mouse IgG coupled to R-phycoerythrin for 30 min at 4 °C, and 5 × 10³ cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Confocal Immunofluorescence Microscopy-- One day post-transfection, 293T cells were seeded in eight-well chamber slides (Lab-Tek) at 3 × 10⁴ cells/well and cultured for 18-36 h at 37 °C. Cells were washed twice with PBS, fixed for 10 min at room temperature in freshly prepared 2% paraformaldhyde in PBS (pH 7.0), washed twice with PBS, and then permeabilized in methanol for 2 min at room temperature. Cells were washed in PBS and then blocked for 10 min at room temperature in PBS containing 3% BSA. Polyclonal goat anti-LTR IgG (28), diluted to a final concentration of 20 µg/ml, rat anti-HveA (1:500 dilution) and mouse anti-FLAG-M2 to detect TRAF3, were diluted in PBS containing 3% BSA and 0.2% Triton X-100 (PBS/BSA/Triton). Primary antibodies were added to the wells to a final volume of 120 µl/well and incubated in a humidified chamber at room temperature for 1 h. Wells were then washed three times in PBS/BSA/Triton buffer. FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories), in combination with Texas Red-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) or donkey anti-rat IgG-Texas Red, was diluted to a final concentration of 1:200 in PBS/BSA/Triton in a final volume of 120 µl/well. Slides were incubated in a humidified chamber at room temperature in the dark for 1 h and then washed three times in PBS/BSA/Triton. The slides were mounted in 80% glycerol in PBS, sealed, and kept at 4 °C in the dark for 1-7 days before visualization. Cells were observed with a Bio-Rad MRC-1024 confocal microscope with a krypton/argon ion laser and a × 60 Nikon objective. Images were acquired using the LaserSharp operation system and were analyzed and manipulated in Adobe PhotoShop. Empty vector-transfected cells or cells stained with normal goat serum or mouse isotype-specific IgG were used for negative controls. Neither control exhibited background staining. Representative staining patterns were based on counting 200 cells.

Co-immunoprecipitations-- 293T cells individually transfected with FLAG-tagged TRAF2, -3, or -5 alone or co-transfected with either HveA or LTR were washed once with PBS and then lysed with 600 µl of lysis buffer (50 mM Tris, pH 9, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Insoluble material was removed by centrifugation at 14,000 rpm for 3 min. Goat anti-HveA or goat anti-LTR was added to the lysates at a final concentration of 5 µg/ml, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were added and mixed at 4 °C for 1 h. The beads were washed, and proteins were solubilized in SDS-PAGE sample buffer. Whole cell lysates were prepared by direct extraction of cell pellets into heated SDS-PAGE sample buffer and clarification by centrifugation (1500 × g for 10 min). Proteins were separated by SDS-PAGE and analyzed by blotting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of LIGHTt66 and Receptor-binding Mutants-- In order to investigate cellular responses initiated by LIGHT, a soluble form (LIGHTt66) was engineered by truncation of the N-terminal 65 amino acids, which removes the cytoplasmic and transmembrane domains. An N-terminal FLAG epitope was added for detection and purification of the cytokine. This construct was expressed in HEK293 cells and purified to homogeneity by ion exchange and immunoaffinity chromatography. The final yield of protein was 80%, and the purity was >99% (Fig. 1A). Soluble LIGHTt66 behaved as an oligomer in SDS-PAGE following treatment with bifunctional cross-linkers (Fig. 1B). Additionally, LIGHTt66 eluted in native gel filtration chromatography as a single narrow peak with a molecular mass of ~76 kDa, which is consistent with a stable trimeric structure, characteristic of this family of cytokines. LIGHT contains two cysteines in the extracellular domain at positions 154 and 187; however, no evidence was found that a disulfide bond was necessary for the formation or stability of the trimer.

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Purification and biochemical characterization of LIGHTt66 and its mutants. A, purification of soluble LIGHTt66-FLAG from HEK293 cells. Top, samples (20 µl) from each purification step were loaded and analyzed by SDS-PAGE (15%) and stained with Coomassie Blue. Bottom, Western blot of each purification step (20-µl samples) detected with anti-FLAG (M2). Relative intensity of the bands agreed with ELISA data indicating that LIGHTt66-FLAG was concentrated 30× after the ion exchange procedure and 100× after affinity purification. Final yield was 60-80% of starting material. B, trimeric structure of LIGHTt66-FLAG. LIGHTt66 (10 µg/ml) was treated with glutaraldehyde (0.1%) or BSOCOES (5 nM) for 30 min at 4 °C, and the reaction was stopped by the addition of Tris (20 mM, pH 8.0). Samples (200 ng) were analyzed by Western blotting using anti-FLAG M2 mAb. Lane 1, untreated LIGHTt66-FLAG; lane 2, treated with glutaraldehyde; lane 3, BSOCOES. C, purification of LIGHTt66 mutants. Top, mutants of LIGHTt66-FLAG produced using 293T cells were affinity-purified from tissue culture supernatant using monoclonal anti-FLAG M2 mAb. Purified protein (100 ng) was analyzed by SDS-PAGE, and proteins were detected by silver stain. Bottom, samples (100 ng) of purified LIGHTt66 mutants were analyzed by Western blot and detected with anti-FLAG M2 mAb. D, gel filtration analysis of LIGHTt66. LIGHTt66-FLAG and mutants were analyzed by FPLC gel filtration on a Superose 12 column at a flow rate of 0.5 ml/min in PBS, and 0.5-ml fractions were collected. Top, fractions from gel filtration of LIGHTt66-FLAG were analyzed by ELISA using HveA-Fc as capture molecule and anti-FLAG M2 mAb as the detecting antibody. Inset, plot of log Mr of calibration proteins versus elution volume. Bottom, samples (1 µl) from gel filtration fractions of LIGHTt66-FLAG and mutants were spotted on a Immobilon membrane (1 µl) and detected with anti-FLAG M2.

Purified LIGHTt66 was active at inducing the death of adenocarcinoma HT29.14s cells with comparable efficiency to LT₁₂ (Fig. 2A). Cytotoxicity was dependent on IFN- (Fig. 2B), as is characteristic of this cell line for induction of apoptosis by LT₁₂, TNF, Fas ligand, and TRAIL, and was maximal after 72 h (data not shown). Over a range of experiments, 50% loss of cell viability was achieved with doses of 10-100 pM LIGHT. LIGHTt66-induced death was blocked in a dose-dependent manner by receptor, LTR-Fc, or HveA-Fc; however, a combination of Fas-Fc, TNFR1-Fc, and TRAILR2-Fc did not inhibit death in this system (Fig. 2C). This latter observation demonstrates specificity of the Fc fusion proteins but also indicates that the death inducing activity of LIGHT is not dependent on induction of a secondary mediator, such as TNF, which was recently shown to occur when CD40 or TNFR2 are activated to induce cell death (22).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   LIGHTt66 is cytotoxic to HT29 cells. A, HT29 cells were incubated with serial dilutions of LIGHTt66, LT12, or TNF in the presence of IFN- (80 units/ml). After a 72-h incubation at 37 °C, cell viability was assessed by MTT dye reduction. B, LIGHTt66 cytotoxicity is dependent on IFN-. HT29 cells were incubated with serial dilutions of LIGHTt66 in the presence or absence of IFN- (80 units/ml), and an MTT dye reduction assay was performed after a 72-h incubation at 37 °C. C, LIGHTt66 cytotoxicity is blocked by co-incubation with LTR-Fc and HveA-Fc. LIGHTt66 (200 pM) was preincubated with varying dilutions of LTR-Fc or HveA-Fc or an equal mixture of Fas-Fc, TNFR1-Fc, and TRAIL R-Fc for 30 min before the addition to HT29 cells in the presence of IFN-. Cell viability was assessed by MTT dye reduction after 72 h. The data in all three panels represents the mean ± S.D. of triplicate wells, and the results are representative of several different experiments.

A three-dimensional model of LIGHT based on the crystallographic structure of LT and TNF was generated to predict residues that are likely to be involved in receptor binding (42) (Fig. 3A). The primary receptor-binding residues in LT are located in the connecting loops of the A  A" and the D  E -strands, which are located on opposite sides of the LIGHT subunit. The receptor binding site, located at the cleft formed between adjacent subunits, is formed as a composite of residues contributed by the A  A" loop from one subunit and the D  E loop of the neighboring subunit. Residues within these loops are not conserved between LIGHT and LT, although significant conservation is apparent in the -strands preceding and following these loops (Fig. 3A). To determine if these loops contained the receptor binding residues, four single amino acid substitutions were introduced into LIGHTt66 at Y173F in the D  E loop or at Q117T, G119E, and L120Q in the A'  A" loop. The mutants were expressed in 293T cells and purified (Fig. 1C). All mutants eluted coincident with wild type LIGHTt66 in gel filtration chromatography, indicating that the mutations do not affect trimer formation (Fig. 1D). LIGHTt66 bound to HveA and LTR with near equal affinity as detected by ELISA assay or surface plasmon resonance (Fig. 3, B and C). The Y173F mutant is the analog of the Y108F mutant of LT, which causes dramatic loss of binding to both TNFRs and HveA (6, 43, 44). This residue is conserved in most TNF-related ligands, and, as expected, when the Y173F mutation was introduced the protein exhibited lower binding affinity to LTR (8-10-fold) and HveA (40-fold) as measured by competitive ELISA or Biacore (Fig. 3, B and C). The lower affinity was due to increased dissociation rates with both receptors (Table I). Q117T, G119E, and L120Q all lie within the A-A" loops, which diverge between LIGHT and LT and LT (Fig. 3A). One or more of these mutants was therefore likely to show altered binding to LTR, while retaining HveA binding. G119E showed reduced but significant affinity for HveA but undetectable binding to LTR. The reduction in affinity of G119E for HveA-Fc was due to an increase in dissociation rate (k_d = 2.0 ± 0.4 × 10³ s¹) (Table I). L120Q and Q117T bound HveA-Fc and LTR-Fc with comparable affinity to LIGHTt66; however, Q117T bound both HveA-Fc and LTR-Fc with similar association rates to LIGHTt66 but slower dissociation rates, so that affinity of this mutant for the receptors was increased relative to LIGHTt66.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Receptor binding characteristics of LIGHTt66 and its mutants. A, molecular model of LIGHT. The theoretical LIGHT model was generated by SwissModel and encompasses amino acids Ser103 to Val240. Upper left panel, -carbon backbone of a LIGHT subunit showing the location of Gln117, Gly119, and Leu120 in the A'  A loop (red) and Tyr173 in the D  E loop (blue); upper right panel, a putative receptor binding face of the LIGHT trimer showing locations of the Gln117, Gly119, and Leu120 in the A'  A loop (red) and Tyr173 in the D  E loop (blue); lower panel, sequence alignment of the A'  A" and D  E regions of LIGHT, LT, and LT (ClustalW, MacVector 6.5). Conserved residues are boxed, and identical residues are shaded. B, LIGHT ELISA. FLAG-tagged LIGHTt66 and its mutants were analyzed by ELISA using the plate-bound LTR-Fc or HveA-Fc as capture molecule and M2 anti-FLAG as detecting antibody, followed by goat anti-mouse horseradish peroxidase. C, surface plasmon resonance. Representative sensorgrams for the binding of LIGHTt66 and its mutants to HveA-Fc and LTR-Fc at a ligand concentration of 300 nM.

LIGHT-induced Cell Death and Integrin Expression Is Mediated by the LTR-- The LIGHTt66 mutants were investigated for their potential to discriminate between LTR and HveA signaling in the HT29.14s cell death assay and induction of ICAM-1 on normal dermal fibroblasts. In the presence of IFN-, L120Q, and Q117T were cytotoxic to HT29 cells with comparable efficiency to LIGHTt66 (Fig. 4A). Y173F, which binds with reduced affinity to both LTR and HveA, was proportionally less effective at inducing cell death, whereas G119E, which has higher affinity for HveA than Y173F, but fails to bind LTR, showed no significant cytotoxicity. This result suggests that LIGHT binding to LTR is required for cell death induction. The induction of ICAM-1 on NHDF by LIGHTt66, although less efficacious than LT₁₂, was lost by mutation of G119E; however, the L120Q and Q117T were as active as wild type, and Y173F was partially active at inducing ICAM-1 at increased concentrations.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of LIGHTt66 mutants on cell death and induction of ICAM. A, cell death. HT29.14s cells were incubated with serial dilutions of LIGHTt66 or the mutants in the presence of IFN-, and cell viability was assessed by MTT dye reduction after 72 h. The data represent the mean ± S.D. of triplicate wells, and this result is representative of five separate experiments. B, ICAM-1 expression. NHDF cells (6 × 104 well) were incubated with the indicated concentrations of cytokine tissue culture medium. After a 36-h, incubation cells were analyzed by fluorescence-activated cell scanning with mAb P2A4 to determine the surface levels of ICAM-1. The -fold induction represents the ratio of the specific fluorescence of the cytokine-treated cells to cells in medium alone.

HT29.14s cells express both LTR and HveA on the cell surface as revealed by flow cytometry, whereas NHDF express significant LTR but little if any HveA (Fig. 5A). The mouse anti-HveA mAb, CW8, but not CW1, blocked LIGHTt66 binding to HveA-Fc, whereas anti-LTR mAb BDA8, but not CDH10, blocked LIGHT binding to LTR (Fig. 5B). Both anti-HveA and anti-LTR polyclonal antibodies effectively blocked LIGHTt66 binding to their respective receptors (Fig. 5B). The monoclonal anti-LTR antibody BDA8 has been shown to inhibit LT₁₂-mediated apoptosis of HT29 cells, whereas CDH10 has been shown to enhance this effect (16). We anticipated that these antibodies should affect LIGHT-mediated apoptosis in the same way. Goat polyclonal anti-LTR induced death of HT29 cells in the presence of IFN- (Fig. 5C), whereas goat polyclonal anti-HveA did not affect cell viability, adding evidence that HveA does not induce apoptosis in this cell line. Further, polyclonal anti-HveA neither enhanced nor inhibited the cell death induced by polyclonal anti-LTR (Fig. 5C). Preincubation with polyclonal goat anti-LTR markedly enhanced the sensitivity of HT29.14s cells to LIGHT-mediated killing (Fig. 5D). Anti-LTR CDH10, which enhances cell death by LT₁₂, also enhanced susceptibility to LIGHTt66. However, preincubation with anti-LTR mAb BDA8, which blocks killing by LT₁₂, resulted in reduced LIGHT-mediated cytotoxicity (Fig. 5D). Preincubation of the cells with polyclonal goat anti-HveA or with the monoclonal anti-HveA antibodies CW1 and CW8 had no effect LIGHT killing.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Effect of anti-LTR and anti-HveA antibodies on HT29 cells. A, HT29.14s cells co-express HveA and LTR. Cells were incubated with anti-LTR (BDA8) or anti-HveA (CW1) (5 µg/ml) (filled area) or normal mouse IgG (dashed line) for 30 min at 4 °C, followed by goat anti-mouse phycoerythrin and analyzed by flow cytometry. FI, fluorescence intensity. B, effect of anti-HveA antibodies on binding of LIGHT to receptors. Left, HveA-Fc-coated wells (3 µg/ml) were incubated with varying concentrations of goat polyclonal anti-HveA or the anti-HveA mAb CW1 or CW8 for 30 min, and then LIGHT was added to a final concentration of 0.25 nM for 1 h before washing and then detection of bound LIGHT with anti-FLAG (M2) and goat anti-mouse IgG-horseradish peroxidase. Right, LTR-Fc-coated wells (3 µg/ml) and incubated with goat polyclonal anti-LTR or the monoclonal anti-LTR BDA8 or CDH10, and then LIGHT was added to a final concentration of 0.25 nM for 1 h. Bound LIGHT was detected as in B. C, effect of antibody cross-linking of HveA and LTR on growth of HT29 cells. HT29.14s cells were incubated with varying concentrations of polyclonal anti-HveA, polyclonal anti-LTR, or a mixture of the two. MTT dye reduction was performed after 72 h. D, effect of polyclonal anti-HveA and anti-LTR on LIGHT-mediated cytotoxicity. HT29 cells were incubated with 10 µg/ml of goat polyclonal anti-HveA, goat polyclonal anti-LTR, or the indicated monoclonal antibodies for 10 min before the addition of LIGHT (0.25 nM). MTT assay was performed after 72 h in culture. Each data point is the mean of triplicate wells, and this result is representative of three experiments.

Collectively, these data indicate that antibody-induced cross-linking of HveA does not induce apoptosis, that HveA signaling does not cooperate with LTR signaling to induce cell death, and that HveA signaling does not trigger protective events sufficient to interfere with the LTR-dependent apoptotic pathway.

Differential Association of TRAF3 with LTR and HveA-- Previous reports have shown that TRAF3 is required for LTR to signal cell death in HT29.14s cells (28-30). An N-terminal truncated mutant of TRAF3, 1-339, when transduced by retrovirus into HT29.14s cells ablated the cytotoxic effect of LIGHTt66 (Fig. 6). This result suggests that the difference in LTR and HveA signaling may reside in the ability to interact with TRAFs. Previous in vitro binding analysis yielded equivocal results concerning HveA interactions with TRAF3 (31, 32). To minimize potential artifacts, we used an in situ association assay to examine TRAF-receptor interactions. 293T cells were co-transfected with TRAF2, -3, or -5 and either HveA or LTR. The proteins were then visualized by confocal microscopy with the assumption that associated proteins will colocalize. All of the TRAFs exhibited a diffuse cytoplasmic staining pattern in the absence of receptors (Fig. 7, a-d). HveA localized to the cellular periphery (Fig. 7e), whereas LTR accumulates in large perinuclear compartments (Fig. 7i). HveA induced redistribution of TRAF2 (7f) and TRAF5 (7h) but not TRAF3 (7g). Indeed, HveA and TRAF5 redistributed into numerous vesicles in a pattern not observed with TRAF2. By contrast, LTR co-localized with all three TRAFs into large perinuclear compartments (Fig. 7, j, k, and l). These results suggest that the inability of HveA to mediate death in this model is due to a lack of interaction with TRAF3.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Dominant negative TRAF3 inhibits LIGHT-induced cell death. T3 TRAF31-339 retrovirus transduced HT29.14s cells and empty vector control were treated with graded concentrations of LIGHTt66 and IFN- (80 units/ml). MTT dye reduction assay was performed after 72 h.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   LIGHT-mediated killing of HT29 cells is dependent on recruitment of TRAF3 to LTR. Co-localization of TRAFs and receptors is shown. 293 T cells overexpressing the indicated molecules as follows. b, f, and j, TRAF2-FLAG; c, g, and k, TRAF3-FLAG; d, h, and l, TRAF5-FLAG; a-d, co-transfected with the empty vector pBABE; e-h, co-expressing HveA; i-l, co-expressing LTR. FLAG-tagged TRAFs were visualized with FITC. LTR and HveA were visualized using Texas Red. Co-localized proteins appear yellow. HveA co-localized with TRAF2 and -5 but not TRAF3. LTR co-localized with TRAF2, -3, and -5.

In co-immunoprecipitation experiments (Fig. 8), both HveA and LTR co-precipitated with TRAF2, -3, and -5 in detergent extracts of cells overexpressing these proteins. This is in contrast to the data presented in Fig. 7, which demonstrate that HveA does not co-localize with TRAF 3 intracellularly, but in agreement with the data of Marsters et al. (31), who reported that a glutathione S-transferase fusion protein of the cytoplasmic domain of HveA precipitated TRAF3 as well as TRAF2 and -5 from HEK293 cells overexpressing these proteins. Collectively, these data indicate that HveA has the potential to interact with TRAF3 under certain conditions but that this interaction does not occur in intact cells, which may reflect distinct compartmentalization routes taken by these receptors. Our data suggest that the inability of HveA to mediate death in this model is due to a lack of intracellular interaction with TRAF3.

View larger version (63K): [in this window] [in a new window]   Fig. 8.   Co-immunoprecipitation of TRAFs with HveA and LTR. 293T cells were transfected with FLAG- tagged TRAF2, -3, or -5 or vector control (pBABE) or were co-transfected with LTR or HveA. A, lysates were prepared and subjected to immunoprecipitation. TRAFs co-precipitated with receptors were detected by Western blot analysis using monoclonal anti-FLAG (M2). Polyclonal anti-HveA was used for immunoprecipitation of the empty pBABE vector as a control. B, in order to determine the total cellular content of transfected TRAFs, equivalent cell numbers of each lysate were loaded in each lane and FLAG-tagged TRAFs were detected by Western blot using M2 (anti-FLAG).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The work presented here demonstrates that LIGHT-LTR system is sufficient to mediate apoptosis in the HT29.14s cell line. Although LTR and HveA are coexpressed on the HT29.14s cells, no role for HveA, either as a direct death-inducing receptor or as a cooperating factor with LTR, could be defined in this cell model. Furthermore, our results demonstrate that LTR-induced death is independent of TNF, TRAIL, or Fas ligand, indicating that LTR signaling may directly activate apoptotic pathways. LTR-mediated cell death signaling by LIGHT was dependent on TRAF3, as is death induced by LT₁₂, indicating their functional similarity.

The molecular structure of LIGHT, based on TNF/LT crystallography, is predicted to be an anti-parallel -sandwich with the propensity to form homotrimers, which is supported by experimental results here and by others (33). Structurally, LIGHT is most homologous to LT and indeed could represent the functional homotrimer that LT is unable to form, since LT is functional only when complexed with LT into heterotrimers (44). This model also reveals a disulfide bond between Cys¹⁵⁴ and Cys¹⁸⁷, located near the top of the trimer; however, we found no change in the protein mobility when analyzed under oxidized or reduced conditions in SDS-PAGE, indicating that the putative disulfide bond is likely to be intramolecular. Mutational analysis revealed specific residues in the A'  A" and D  E loops, which contributed significantly to the ability of LIGHT to interact with its receptors. The identification of residues that can selectively alter LIGHT binding to one of its receptors should prove valuable in deciphering signaling pathways responsible for the cellular responses activated by this ligand.

One of the more striking effects of LIGHT is its cytotoxic effect on several tumor cell lines in culture and its ability to prevent tumor formation in mice (33, 34). The relative importance of the two cellular receptors, HveA and LTR, for induction of death in tissue culture has not been directly addressed. Zhai et al. (34) examined the effect of soluble LIGHT on several cell lines and reported that one cell line, PC3, which expresses LTR in the absence of HveA, is resistant to LIGHT-mediated cytotoxicity. The authors concluded that both receptors are necessary for LIGHT-mediated cytotoxicity. However, they did not examine the possible activation of other mechanisms of resistance, such as induction of protective cellular responses by NF-B (45), or expression of LIGHT-inhibitory molecules, such as the recently described soluble decoy receptor DCR3, which binds both LIGHT and Fas ligand (46, 47). The ability of LTR to induce apoptosis, in response to LT₁₂ or antibody cross-linking, is well established (16), whereas cross-linking of HveA was not sufficient to induce death in cells susceptible to LIGHT-induced death. The inability of antibodies to block death induced by LIGHTt66 yet inhibit the binding of this ligand to HveA further strengthens the conclusion that HveA is not a direct apoptosis-inducing receptor. We addressed the possibility that HveA might cooperate in LTR-mediated cell killing by a ligand-independent mechanism; however, anti-HveA antibodies had no effect on anti-LTR-mediated cytotoxicity. Finally, LIGHTQ117T, which binds HveA but not LTR, was inactive in inducing death of HT29.14s cells. Collectively, these data establish that engagement of HveA neither enhances nor inhibits LTR-mediated cytotoxicity in response to LIGHT binding. Our results do not support a cooperative model for death signaling but rather reinforce a model where the LTR is necessary and sufficient to induce cell death. LTR was also sufficient to induce activation of ICAM-1 expression. By comparing the effect of the HveA-specific mutant G119E with that of Y173F, which binds HveA with lower affinity than G119E but retains LTR binding, we showed that up-regulation of ICAM by NHDF cells is also induced by LIGHT in a HveA-independent process. This is not surprising, because HveA expression on NHDF is near the detection limit by fluorescence-activated cell scanning and thus may be nonfunctional. We do note that LT₁₂ was more potent than LIGHT in the induction of ICAM-1 (5).

LTR-mediated cell death depends on recruitment of TRAF3 (28). HveA co-localized with TRAF2 and -5, which are not involved in signaling apoptosis, but not TRAF3. By contrast, LTR co-localized with TRAF2, -3, and -5 into characteristic perinuclear compartments. These data are consistent with those of Hsu et al. (32), who, using yeast two-hybrid screening, demonstrated that the cytoplasmic domain of HveA interacts with TRAF2 and -5 but not with TRAF3. In contrast, HveA, like LTR, co-precipitated with all three TRAFs from detergent extracts of 293T cells overexpressing these proteins. Our immunoprecipitation data are in agreement with those obtained by Marsters et al. (31) using a glutathione S-transferase fusion protein containing the cytoplasmic domain of HveA. Collectively, these data indicate that HveA can interact with TRAF3 in experimental systems but that in the intracellular environment this interaction is either absent or insufficient to mediate biological effects in untransfected cells. HveA and TRAF3 may be present in the cell in different subcellular compartments and thus prevented from interaction.

The resistance to LIGHT-mediated apoptosis of HT29.14s cells expressing TRAF3 dominant negative mutant further strengthens our hypothesis that LTR signaling is responsible for the death-inducing activity of LIGHT. The shared ligand specificity of LTR and HveA and the binding of common signaling proteins, TRAF2 and -5, makes it tempting to speculate that cooperative signaling may occur in appropriate cell types. Perhaps activation of NF-B by TRAF2 or -5 via HveA and LTR signaling simultaneously in the same cell type may enhance NF-B-driven responses.

Although both engage LTR, the phenotype of LT- and LT-deficient mice establishes that LT₁₂ and LIGHT are not redundant. Mice rendered deficient in LT and LT demonstrate the essential roles played by the LT-LTR system in the development of lymphoid tissues. Failure of lymph node genesis results from ablation of both LT (48, 49) and LT (11, 12, 50). LTR/ and LT/ mice share other phenotypes including the absence of colon-associated lymphoid tissues, loss of E7^high integrin+ T cells, disorganized splenic architecture, loss of splenic marginal zones, abnormal T/B cell segregation, and absence of follicular dendritic cell networks. In contrast to TNF receptor p55/ mice, antibody affinity maturation was impaired. The fact that LTR/ mice exhibit distinct defects when compared with LT/ and LT/ mice (e.g. aberrant localization of PNA⁺ cell clusters around central arterioles) implies that other ligands may be able to activate LTR. Here LIGHT becomes a lead candidate (6).

Further, the cellular expression patterns of HveA and LTR are distinct; HveA is prominent on lymphoid cells, where LTR is noticeably absent (51). Additionally, the activation signals required for expression of LIGHT and LT by T cells are different (6). LIGHT expression requires stimulation with both PMA and calcium ionophore, whereas LT₁₂ expression requires only PMA, suggesting that these ligands are important for different T cell functions. Collectively, these observations indicate that the LIGHT-HveA system will activate physiologic functions distinct from the LT-LTR system.

	ACKNOWLEDGEMENTS

We thank Jeff Browning for cytokines; Jurg Tschopp for the TRAIL R2-Fc protein; Hiroyasu Nakano, George Mosialos, and Elliott Kieff for the tagged TRAFs; and Cheryl McLaughlin for the excellent assistance with figures and manuscript preparation.

	FOOTNOTES

^* This work was supported in part by U. S. Public Health Service, National Institutes of Health Grants CA69381, AI03368 (to C. F. W.), and NS-36731 (to R. J. E.), American Cancer Society Grant IM663 (to C. F. W), and fellowships from National Institutes of Health Grants T32AI07469 (to A. A. G.) and AG00252 (to C. A. B.). This is publication 332 from the La Jolla Institute for Allergy and Immunology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 858-558-3500; Fax: 858-558-3525; E-mail: carl_ware@liai.org.

² J. C. Whitbeck, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; LT, lymphotoxin; LTR, LT- receptor; HveA, herpesvirus entry mediator; ICAM, intercellular adhesion molecule; IFN-, interferon-; mAb, monoclonal antibody; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHDF, normal human dermal fibroblast; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TRAF, TNF receptor-associated factor; TNFR, TNF receptor; NF-B, nuclear factor B; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography; BSOCOES, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone.

	REFERENCES

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