Proteolysis Generated by Alternate Splicing Or Chromosome 19P13.3

Genomic Characterization of LIGHT Reveals Linkage to an Immune Response Locus on Chromosome 19p13.3 and Distinct Isoforms Generated by Alternate Splicing or This information is current as Proteolysis of September 23, 2021. Steve W. Granger, Kris D. Butrovich, Pantea Houshmand, Wilson R. Edwards and Carl F. Ware J Immunol 2001; 167:5122-5128; ; doi: 10.4049/jimmunol.167.9.5122 Downloaded from http://www.jimmunol.org/content/167/9/5122

References This article cites 34 articles, 14 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/167/9/5122.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Genomic Characterization of LIGHT Reveals Linkage to an Immune Response Locus on Chromosome 19p13.3 and Distinct Isoforms Generated by Alternate Splicing or Proteolysis1,2

Steve W. Granger, Kris D. Butrovich, Pantea Houshmand, Wilson R. Edwards, and Carl F. Ware3

LIGHT is a member of the TNF cytokine superfamily that signals through the lymphotoxin (LT)␤ receptor and the herpesvirus entry mediator. LIGHT may function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus, which may provide signiﬁcant selective pressure shaping the evolution of LIGHT. Here, we deﬁne the molecular genetics of the human LIGHT locus, revealing its close linkage to the TNF superfamily members CD27 ligand and 4-1BB ligand, and the third complement protein (C3), which positions LIGHT within the MHC paralog on chromosome 19p13.3. An alternately spliced isoform of Downloaded from LIGHT mRNA that encodes a transmembrane-deleted form is detected in activated T cells and gives rise to a nonglycosylated protein that resides in the cytosol. Furthermore, membrane LIGHT is shed from the cell surface of human 293 T cells. These studies reveal new mechanisms involved in regulating the physical forms and cellular compartmentalization of LIGHT that may contribute to the regulation and biological function of this cytokine. The Journal of Immunology, 2001, 167: 5122Ð5128.

IGHT is a recently identified member of the TNF cyto- ally, inhibition of LIGHT with soluble LT␤R-Fc decoy or anti- http://www.jimmunol.org/ kine superfamily closely related to lymphotoxin (LT)4 ␣ LIGHT Ab suppresses graft vs host disease (10) indicating LIGHT L and LT␤ (1). LIGHT is a type II transmembrane glyco- is involved in effector functions mediated by T cells. protein that is transiently expressed on the surface of activated LIGHT may also function as a direct viral deterrent. HSV in- T lymphocytes (1) and dendritic cells (2). Two differentially fection induces premature death of activated T cells (11) and can expressed cell surface receptors mediate LIGHT signaling, the block maturation of dendritic cells (12, 13) potentially leading to herpesvirus entry mediator (HVEM also known as HveA), localized immune suppression. Envelope glycoprotein D binds prominent on T cells, and the LT␤R, found on stromal cells, but HVEM as one of the cellular entry routes used by HSV (14). Gly- absent on lymphocytes (1). LIGHT interacts with a soluble coprotein D directly competes for the binding of membrane binding protein, decoy receptor 3, which also interacts with Fas LIGHT to HVEM, whereas LIGHT can interfere with HSV entry by guest on September 23, 2021 ligand (FasL) (3, 4). by down-regulation of HVEM (1, 15). These observations suggest The interaction of LIGHT with the LT␤R induces proinflam- that LIGHT may be an integral part of host immunity to herpes- matory gene expression through activation of the transcription fac- virus, which is strongly supported by recent observations on the tor NF-␬B, similar to the LT␣␤ complex, the ligand required for role that LIGHT and LT␣␤ play in resistance to CMV (16). The lymphoid organogenesis and development of NK (5) and NK-T interaction of viral gene products with TNF superfamily (TNFSF) cells (6). LIGHT induces apoptosis in susceptible colon carcinoma members may have shaped the evolutionary course of these mol- cells via the LT␤R (7), and in vivo, transduction of tumor cells ecules, which prompted us to examine the genetic organization of with LIGHT cDNA suppresses tumor outgrowth (8). Evidence is LIGHT (TNFSF14) for insights into the regulation of this cytokine. accumulating that LIGHT signaling through HVEM may function Here, we define the molecular genetics of the human LIGHT as a costimulatory molecule for T cells, including the enhancement genomic locus, revealing close linkage to CD27 ligand (CD27L) of T cell proliferation and secretion of IFN-␥ (2, 9, 10). Addition- (CD70, TNFSF7), 4-1BB ligand (4-1BBL)(TNFSF9), and the third complement protein (C3) defining a novel immune response locus within an MHC-like region on chromosome (Chr) 19p13.3. A Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, detailed analysis of LIGHT gene organization uncovered a San Diego, CA 92121 differentially spliced transcript that encodes a novel transmem- Received for publication June 29, 2001. Accepted for publication August 24, 2001. brane-deleted form of LIGHT, which is nonglycosylated, unlike The costs of publication of this article were defrayed in part by the payment of page the membrane form. Furthermore, membrane LIGHT is shed charges. This article must therefore be hereby marked advertisement in accordance from the surface revealing multiple mechanisms involved in with 18 U.S.C. Section 1734 solely to indicate this fact. regulating the physical form and cellular compartmentalization 1 This work was supported in part by U.S. Public Health Service, National Institutes of LIGHT. of Health Grants CA69381, AI03368, and AI48073. 2 This is publication 428 from the La Jolla Institute for Allergy and Immunology. 3 Address correspondence and reprint requests to Dr. Carl F. Ware, Division of Mo- Materials and Methods lecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Cells, cytokines, and Abs Center Drive, San Diego, CA 92121. E-mail address: [email protected] The human II-23 cell line (D7 subclone), a CD4ϩ T cell hybridoma line 4 Abbreviations used in this paper: LT, lymphotoxin; BAC, bacterial artificial chro- (17), was maintained in RPMI 1640 containing 10% FBS (HyClone Lab- mosome; FasL, Fas ligand; HVEM, herpesvirus entry mediator; TNFSF, TNF super- ␮ family; NP40, Nonidet P-40; Chr, chromosome; PNGase F, peptide-N-glycosidase F; oratories, Logan, UT) and 100 U/ml penicillin/100 g/ml streptomycin CD27L, CD27 ligand; 4-1BBL, 4-1BB ligand; C3, third complement protein; endo H, (Life Technologies, Grand Island, NY). II-23 cells were activated with 100 endoglycosidase H. ng/ml PMA or PMA and 1 ␮g/ml ionomycin for 4 h. Human kidney 293

cells expressing the adenovirus large T Ag (293T) (American Type Culture bromide and photographed under UV trans-illumination with an alpha im- Collection, Manassas, VA) were grown in DMEM containing 10% FBS ager (Alpha Innotech, San Leandro, CA). and antibiotics. Human PBL were isolated by Ficoll gradient centrifugation and adherent cell depletion as described previously (1). B lymphocytes Transfections were removed by passage through nylon wool, and the T lymphocytes were activated with anti-CD3 (OKT3) (1 ␮g/ml) and anti-CD28 (1 ␮g/ml) (BD Human embryonic kidney 293T cells, in six-well dishes (50% conﬂuent), ␮ Biosciences, Mountain View, CA) and cultured in RPMI 1640/FBS with were transfected with 3 g of cDNA per well, using the calcium phosphate IL-2 (10 ng/ml). After 7 days of culture, these T cells were activated with coprecipitation method (22). The cells were incubated with the precipitate PMA (100 ng) and ionomycin (1 ␮g) in fresh medium at 2 ϫ 106 cells/ml for 12 h, and then fresh complete medium was added for 48 h to achieve as described previously (1). Rat anti-human LIGHT was prepared by im- maximal protein expression. munization with 50 ␮g of puriﬁed LIGHTt66, a recombinant truncated form with a deletion of the cytoplasmic and transmembrane regions gen- Receptor-mediated ligand precipitation and Western blot erating a soluble protein as previously described (7). Anti-human LIGHT analysis

Omniclone is a bacterially expressed combinatorial Ab containing VH and ␬ Tissue culture supernatants were harvested from transfected 293T cells, V -chains generated from a BALB/c mouse immunized with recombinant ϫ soluble human LIGHTt66 (Biosite Diagnostics, San Diego, CA) (18). This and debris was removed by centrifugation at 1000 g for 5 min. Super- natants were then treated with a protease inhibitor mixture (1 mM PMSF, Ab reacts with natural and recombinant human LIGHT in ﬂow cytometry ␮ ␮ and immunoprecipitation; no cross-reactivity was observed with TNF, 10 mM iodoacetamide, 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.02% ␣␤ azide) and adjusted to a ﬁnal concentration of 1% Nonidet P-40 (NP40). LT , or mouse LIGHT (a detailed description of the properties of this ϫ 6 reagent is in preparation). Mouse anti-methamphetamine Omniclone was Cellular extracts were prepared by treatment of cell pellets (2.5 10 used as an isotype control and was provided by G. Valkirs (Biosite Diag- cells) with 0.7 ml of 1% NP40 lysis buffer containing 50 mM HEPES, pH nostics). Fc fusion proteins consisting of the ecto domain of HVEM or 7.5, 150 mM NaCl, 20 mM EDTA, 1 mM PMSF, 10 mM iodoacetamide,

␮ ␮ Downloaded from TNFR1 fused to the Fc region of human IgG1 were produced in baculo- 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.02% azide. Supernatants or detergent lysates were precleared by incubation with 10 ␮g/ml human IgG virus expression system and affinity purified as previously described in ␮ detail (1, 19, 20). or mouse isotype control and 30 l of protein G-Sepharose beads (Phar- macia, Peapack, NJ) for2hat4¡C with nutation. Ligands were precipitated RT-PCR analysis from supernatants or lysates by incubation with 10 ␮g/ml HVEM-Fc, 10 ␮g/ml TNFR1-Fc, or 10 ␮g/ml mouse anti-LIGHT Omniclonal Ab for 2 h RNA was isolated from 2 ϫ 106 cells using 1 ml of TRIzol (Life Tech- at 4¡C with protein G-Sepharose beads. Washed immunoprecipitates were nologies) following the manufacturer’s protocol. First-strand cDNA syn- treated with recombinant forms of endoglycosidase H (endo H) and pep- thesis was performed using Superscript II (Life Technologies) as described tide-N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) for http://www.jimmunol.org/ previously (21). PCR amplification, using oligonucleotide primers derived 1hat37¡C. Samples were resolved using a Tris-glycine SDS-polyacryl- from the human LIGHT cDNA sequence (forward 5Ј-TCAGTGTTTGT amide (14%) gel (NOVEX, San Diego, CA), and proteins were trans- GGTGGATGGA-3Ј, reverse 5Ј-CTTCCTTCACACCATGAAAGC-3Ј) ferred to Polyscreen polyvinylidene difluoride transfer membrane was accomplished using the following amplification schedule: 95¡C for 2 (NEN, Boston, MA) using a semidry blotting unit (Fisher, Pittsburgh, Ј min; 30 cycles of 95¡C for 30 s, 58¡C for 30 s, 7¡C for 30 s; and 72¡C for PA). Immune complexes were detected with goat F(ab )2 anti-rat IgG 10 min. Following amplification, products were analyzed by agarose conjugated to HRP and detected by ECL (Pierce SuperSignal; Pierce, (1.5%) gel electrophoresis. The gels were stained using 1 ␮g/ml ethidium Rockford, IL) as described (7). by guest on September 23, 2021

FIGURE 1. A, Chromosomal localization of human LIGHT and the costimulatory locus. The chromosomal location of LIGHT was determined by ﬂuorescence in situ hybridization (FISH) of metaphase chromosomes using hybridization probes including the entire BAC clone containing the LIGHT genomic locus and Chr19-speciﬁc DNA. Cohybridization of each probe, indicated by arrows, on a single chromosome indicated that LIGHT is located at the terminus of the short arm of Chr19. B, Diagram of the 19p13.3 region. C, Organization of the C3, LIGHT, CD27L, 4-1BBL immune response locus. Arrows indicate the transcriptional orientation of each gene, and solid blocks represent exons. LIGHT is separated from C3 by 7.78 kb, from CD27L by ϳ79 kb, whereas CD27L and 4-1BBL are separated by ϳ235 kb. D, Organization of the FasL, GITR ligand, and OX40 ligand genomic locus. FasL is separated from GITRL by 374 kb. GITRL and OX40L are separated by 134 kb. The accession numbers of BAC clones or assembled contigs that contain the various loci are shown above each diagram. E, Phylogenetic analysis of various TNFSF members. Sequences of the extracellular domains were aligned using ClustalW (BLOSUM series matrix), and phylogeny was inferred using the Neighbor joining method and bootstrapped (1000 reps) (MacVector 7.0). The tree is unrooted and numbers indicate percentages of 1000 bootstrap replicates. 5124 MOLECULAR GENETICS OF LIGHT

Cloning, sequencing, and sequence analysis The human LIGHT locus-containing P1 clone was isolated by PCR screening a P1 library using the following LIGHT specific oligonucleotide primers: forward 5Ј-GCTCCTGGGAGCAGCTGATACAA-3Ј and reverse 5Ј- TGGGTTGACCTCGTGAGACCTTCG-3Ј (IncyteGenomics, Palo Alto, CA). The LIGHT genomic locus was sequenced in both 5Ј and 3Ј directions using primers specific for the LIGHT cDNA sequence and cycle sequencing chemistry (Applied Biosystems, Foster City, CA). Sequences were analyzed on an Applied Biosystems prism 310 sequencer and contigs were assembled using the AssemblyLIGN program (MacVector 7.0; Oxford Molecular, Madison, WI). RT-PCR products were cloned using the TA cloning system (Invitrogen, San Diego, CA). The PCR amplicon was digested and ligated into the pCMV2 mammalian expression vector in frame with amino-terminal FLAG coding sequences or pCDNA3.1ϩ. Bacterial artificial chromosome (BAC) DNA was analyzed for gene content using the gene finding algorithm GenScan provided by the Massachusetts Insti- tute of Technology Computational Biology Department (http://genes.mit. edu/GENSCAN.html) (23). Hypothetical proteins were identified by pBLAST search of GenBank. Consensus motifs for sequence-specific DNA binding proteins were identified using the transcription element search system (TESS) at the University of Pennsylvania (Philadelphia, PA) to search the TRANSFAC database (http://www.cbil.upenn.edu/tess/) (24). Downloaded from Results and Discussion The chromosomal location of LIGHT In a BLAST search of the unfinished high throughput genomic sequence database, two BAC sequences (AC008760 and

AC025343) from human Chr19 were identified; each contained the http://www.jimmunol.org/ entire LIGHT gene (TNFSF14). The identity between LIGHT mRNA nucleotide sequences and the Chr19 genomic sequences, with the exception of introns, was absolute (E value of 0.0). This result was puzzling because a prior report assigned LIGHT to Chr16 by in situ hybridization of metaphase chromosomes (8). This discrepancy was resolved by screening a P1 phage library for the LIGHT genomic locus, and the entire P1 DNA clone was used for in situ hybridization of metaphase chromosomes (Fig. 1A). In FIGURE 2. Identification of a novel LIGHT transcript (LIGHT ⌬TM) this analysis, a Chr19 specific probe cohybridized with the P1 expressed in activated II-23 cells and PBL. A, Diagram of LIGHT tran- by guest on September 23, 2021 LIGHT probe and verified that the chromosomal location of scripts with the locations of the oligonucleotide primers used to amplify LIGHT cDNA by RT-PCR. B, LIGHT ⌬TM expression in II-23 cells by LIGHT is 19p13.3. RT-PCR analysis. RNA was isolated from II-23 cells 4 h postactivation. The LIGHT gene resides adjacent to C3 and within a T cell PCR products from the amplification of cDNA from untreated (lane 2)or PMA/ionomycin-treated (lane 3) II-23 cells were analyzed by agarose gel costimulatory locus containing CD27L and 4-1BBL electrophoresis. As controls, RNA from II-23 cells activated with PMA and Identification of the LIGHT-containing BAC clone AC008760 ionomycin without reverse transcriptase (lane 4) or water alone (lane 5). C, yielded 200 kb of genomic DNA for analysis of closely linked LIGHT ⌬TM expression in human peripheral blood T lymphocytes by genes. Sequence analysis of this BAC clone revealed tight linkage RT-PCR analysis. Anti-CD3-activated peripheral blood T cells were grown of the C3 to LIGHT separated by only 7.78 kb with the 3Ј end of in IL-2 for 7 days and placed in fresh IL-2 medium (lane 2) or IL-2 me- C3 adjacent to the 5Ј end of LIGHT (Fig. 1C). Several TNF family dium with PMA and ionomycin for2h(lane 3)or4.5h(lane 4). At these time points RNA was extracted and analyzed for LIGHT expression by members localize in clusters, prompting a screen for related li- RT-PCR as in A above. As controls, RNA without reverse transcriptase gands in this region using the GenScan gene finding algorithm. from cells activated for 4.5 h (lane 5), water alone (lane 6), and 50 ng of The genomic locus for CD27L (CD70/TNFSF7) was identified on LIGHT plasmid (lane 7) were used as templates for PCR. both BAC clones AC008760 and AC025343 and was mapped on NT 011098 to within 79 kb of LIGHT. In addition, the genomic locus of 4-1BBL (TNFSF9) was identified on a BAC clone endothelial growth inhibitor to Chr9. The evolutionary relationship (AC010503) that overlaps BAC AC025343 containing the LIGHT of these paralogs has been the subject of much interest and con- and CD27L genes. 4-1BBL was mapped on NT 011169 to within troversy in understanding the origins of the MHC (28). These para- 235 kb of CD27L. Therefore, C3, LIGHT, CD27L, and 4-1BB-L all logs contain class I, complement and TNF-related genes among reside on 19p13.3 in a region that spans ϳ370 kb (Fig. 1, B and C). other conserved markers. The LIGHT locus is notably reminiscent The close genetic linkage of LIGHT to CD27L and 4-1BBL sup- of the TNF locus containing LT␤, TNF, and LT␣ closely linked to ports the possibility that this gene cluster arose from localized gene C2 and C4 within the MHC (29, 30). It should be noted that within duplication events (25). the complement protein family, C3 displays the highest sequence The positioning on Chr19p13.3 places LIGHT within a large similarity to C4 (31). In addition, LIGHT and LT␤ share function- genetic region paralogous to the MHC on Chr6p21.3, which is ϳ8 ality and the highest sequence similarity within the TNF super Mb in size (26, 27). The Chr19p13.1-p13.3 MHC paralog is one of family and LT␤ resides near the C4 gene. During evolution, gene four regions thought to have arisen by chromosomal duplication, duplication events are often followed by translocations that either which include Chr1q21-q25/p11-p32 and Chr9q33-q34. Other relocate the gene cluster to different chromosomes or break up the TNF-related superfamily members map to these MHC paralogs, cluster altogether. Therefore, it is tempting to speculate that a FasL and OX40 ligand to Chr1, and CD30 ligand and vascular translocation of the TNF-LT locus gave rise to the entire LIGHT The Journal of Immunology 5125

⌬ ⌬

FIGURE 3. LIGHT TM is produced by alternative splicing. Full-length membrane LIGHT transcripts are spliced as indicated on the left. LIGHT TM Downloaded from is produced by the joining of a cryptic splice donor sequence in exon 1 at nucleotide position 112, shown in bold, to the splice acceptor sequence that defines the start of exon 2. The LIGHT ⌬TM transcript encodes a protein that is truncated from amino acid 37Ð73, which results in a deletion of the transmembrane region. locus or vice versa. However, CD27L and 4-1BBL have only three involving the entire gene cluster, presumably occurring during a exons, whereas TNF, LT␤,LT␣, and LIGHT have four exons sug- chromosomal duplication. This result would support the idea that gesting that the LIGHT locus was not directly descendent from the Chr19 and Chr1 MHC paralogs are more closely related to each other http://www.jimmunol.org/ TNF/LT locus. This result compelled an examination of the than the Chr6 paralog. Given the close linkage of LIGHT and C3, genomic organization of TNF-related ligands on Chr1, where suf- which is considered the primordial gene of C5 and C4 based on both ficient information on gene structure could be extracted. The gene phylogenetic and functional arguments (31), LIGHT may be primor- content of the genomic DNA adjacent to the FasL gene, within the dial to LT␤ and FasL. The chromosomal position of the LIGHT locus contig NT 000039, which is comprised of nine P1-derived artificial is conserved in mice; however, insufficient phylogenetic analysis is chromosome clones and two BAC clones of various sizes, con- available for most TNFSF members limiting further analysis. tained FasL (TNFSF6), GITR ligand (TNFSF18), and OX40 li-

gand (TNFSF4) (Fig. 1D). Although the genes in the FasL locus by guest on September 23, 2021 span a greater distance than those of the LIGHT locus, the gene The genomic organization of LIGHT orientation and exon organization of FasL is strikingly similar to The LIGHT gene spans 5.1 kb (from AUG to stop codon) and is the LIGHT locus. Additionally, GITRL and OX40L contain three comprised of four exons with similar organization to FasL, LT␤, exons matching CD27L and 4-1BBL. Phylogenetic analysis of the and other TNF family members (Table I). Exon 1 of LIGHT en- amino acid sequence of the TNF family reveals significant sequence codes the first 73 amino acids of the polypeptide, which comprises similarity in the proteins encoded by LIGHT and FasL loci that the entire cytoplasmic tail (aa 1Ð38), transmembrane domain (aa corresponds with their position in the gene clusters (Fig. 1E). (GITRL 38Ð58), and the beginning of the extracellular stalk region. The does not fit this tree well owing to significant divergence in sequence second and third exons encode amino acids 74Ð85 and 86Ð100, in the initial A ␤ strand.) However, the sequence and organizational respectively, which make up the stalk region and the beginning of similarities do not apply when the LIGHT locus is compared with the the trimerization domain. The fourth exon encodes the remainder LT␣␤/TNF locus. of the trimerization domain, the receptor binding domain (amino At a functional level, FasL and LIGHT exhibit significant amino acids 101Ð240), and includes a site for N-linked glycosylation acid sequence homology in their ectodomains (31%), second only (Asn102). Based on structural homology with LT␣ (32) and sup- to the similarity between LIGHT and LT␤ (34%) (1). Although portive evidence from modeling and biochemical analysis, the tri- binding to distinct cellular receptors, FasL and LIGHT do share the merization domain of LIGHT folds into an anti-parallel ␤ sand- ability to bind soluble DCR3 (4). Although various possible com- wich; wherein the receptor-binding sites are formed from adjacent binations of gene duplication and translocation may explain the subunits (7). origin of each gene cluster, collectively, the relationship between The LIGHT transcript has been previously reported as 2.5 or 2.7 the FasL and LIGHT loci is suggestive of a duplicative event kb by Northern blot analysis, although two considerably smaller

Table I. Intron/exon arrangement of LIGHT

5Ј End 3Ј End Size (bp) Encodes (aa) Size (aa)

Exon 1 ATGG- -GCCT 219 (267 from cap) 1Ð74 73 Intron 1 GTGA- -TCAG 2401 Exon 2 GACG- -CAAG 37 74Ð85 12 Intron 2 GTGA- -CCAG 258 Exon 3 AGCG- -ACAG 42 86Ð99 14 Intron 3 GTGA- -ACAG 1762 Exon 4 GGGC- -AGG stop 425 (827 to polyA) 100Ð240 140 5126 MOLECULAR GENETICS OF LIGHT

by Northern blot analysis. The ﬁnding that CD70, LIGHT, and 4-1BBL all colocalize to a small region on human Chr19 p13.3 supports the possibility that these molecules may be commonly regulated by higher order chromosomal regulation involving chro- matin remodeling. However, each ligand displays a different cellular expression pattern, LIGHT is produced by T cells, whereas CD27L is expressed by B cells and 4-1BBL resides on macro- phages, yet all of their receptors are expressed by T cells, under- scoring their functional roles in T cell activation.

An alternate spliced form of LIGHT RT-PCR analysis of LIGHT expression in the human T cell hybridoma (II-23.D7) revealed a second transcript ϳ100 nt smaller in size than the full-length LIGHT message that was also inducible with PMA and ionomycin treatment (Fig. 2). DNA sequencing revealed that the smaller transcript contained an internal deletion of 36 amino acids that removes the entire transmembrane domain, referred to as LIGHT ⌬TM (accession number AY028261). LIGHT ⌬TM transcripts were observed at levels considerably Downloaded from lower than the full-length LIGHT transcript (Fig. 2B). A similar pattern of inducible expression was also observed in human PBLs following activation with PMA and ionomycin (Fig. 2C). The detection of LIGHT ⌬TM in activated peripheral blood T lymphocytes is supportive of a possible biological role for this isoform of

LIGHT in vivo. http://www.jimmunol.org/ Analysis of the genomic sequence of LIGHT, using a splice donor and acceptor consensus site prediction model, revealed a splice donor consensus sequence at the exact nucleotide corre- FIGURE 4. Analysis of LIGHT ⌬TM expression in 293T cells. A, sponding to the deletion in LIGHT ⌬ TM. Therefore, this alterna- LIGHT ⌬TM binds HVEM but is not secreted. 293T cells were transiently tive transcript is generated by joining the cryptic splice donor in transfected with cDNA encoding LIGHT ⌬TM, soluble FLAG-tagged exon 1, at nucleotide position 111, to the splice acceptor that de- LIGHT (LIGHTt66) containing the VCAM signal peptide for secretion, or ϩ fines the beginning of exon 2, at nucleotide position 218, resulting empty pCDNA3.1 . Spent culture supernatants and nonionic detergent ly- in the removal of 107 nucleotides including the transmembrane by guest on September 23, 2021 sates of the cells were precleared with human IgG and then treated with domain in exon 1 (Fig. 3). Thus, direct genetic evidence from human HVEM-Fc or TNFR1-Fc and protein G beads. The immobilized splice donor-acceptor prediction algorithms supports alternative ligands were resolved on SDS-PAGE and analyzed by Western blot using a rat anti-LIGHT polyclonal serum for detection. Immunoprecipitates from cells transfected with LIGHT ⌬TM (lanes 3, 6, and 7), soluble LIGHTt66 (lanes 1, 4, and 9), or empty pCDNA3.1ϩ (lanes 2, 5, and 8). Purified soluble LIGHTt66 (10 ng) was included (lane 10) as a size standard. De- tergent lysate (5 ϫ 104 cell equivalents) from each cell was analyzed to assess relative amount of expressed protein (lanes 7–9). B, LIGHT ⌬TM is not glycosylated. Immunoprecipitates from the NP40 cell lysates of 293T cells transfected with soluble LIGHT ⌬TM (lanes 1–3), pCDNA3.1ϩ alone (lane 4–6), LIGHTt66 (lanes 7–9), or full-length LIGHT (lanes 11–13) encoding plasmids are shown. Detergent lysates were precleared with an isotype control and then immunoprecipitated with mouse anti-human LIGHT Ab and protein G. The immunoprecipitates were digested with either endo H (lanes 2, 5, 8, and 12) or PNGaseF (lanes 3, 6, 9, and 13), or were left untreated (lanes 1, 4, 7, and 11); purified soluble LIGHT (lane 10).

cDNAs have been cloned of 1169 nt (1) and 1491 nt (9). There- fore, it is likely that the full-length transcript has yet to be defined. Analysis of the nucleotide sequences 5Ј of the LIGHT gene up to the 3Ј region of the C3 gene revealed three potential consensus FIGURE 5. LIGHT is shed from the surface of 293T cells. 293T cells TATAA elements at nucleotide positions Ϫ1979, Ϫ1762, and were transiently transfected with plasmids encoding either membrane ϩ Ϫ839 relative to the ATG of the first exon. In addition, a combi- LIGHT or empty pCDNA3.1 . HVEM-Fc was used to precipitate LIGHT nation of consensus motifs for sequence-specific DNA binding from the tissue culture supernatant (lane 1) or cell lysate (lane 3) of membrane LIGHT-transfected 293T cells. The precipitates were resolved by proteins, consistent with the inducibility of LIGHT by TCR sig- SDS-PAGE and Western blot analysis using a rat anti-LIGHT polyclonal naling, are also present. These motifs, among others, include AP-1, serum as probe. TNFR1-Fc was used to control for nonspecific binding to ␬ NF- B, and Oct-1 binding sites, which are all present in the highly membrane LIGHT (lane 2) or supernatants (lane 4). An equivalent amount TCR-inducible IL-2 promoter (33). Usage of the TATAA element (5 ϫ 104 cell equivalents) from each transfected cell lysate was loaded in at nucleotide position Ϫ1762 would yield transcripts of ϳ2.8 kb; lanes 5 and 7. Purified recombinant soluble LIGHTt66 (10 ng) (lane 6) this transcript size would be more consistent with those observed used as a molecular mass marker. The Journal of Immunology 5127 splicing of LIGHT as the mechanism for the generation of the brane, as occurs for TNF (35). The shed form of LIGHT binds smaller LIGHT transcript detected by RT-PCR. HVEM-Fc suggesting that it is a functional ligand. However, with A cDNA encoding LIGHT ⌬TM was cloned into the mamma- FasL the soluble form generated by shedding is not capable of lian expression vector (pCDNA3.1ϩ) to study its biologic activity. inducing apoptosis (34). Bearing on this result, recombinant 239T cells transfected with this construct expressed a 28-kDa pro- LIGHT truncated near the cleavage site is relatively unstable com- tein that reacts with a polyclonal rat anti-human LIGHT antiserum pared with the LIGHTt66 form (C. Ware, unpublished observa- by Western blot analysis (Fig. 4A). In addition, HVEM-Fc fusion tions), suggesting the possibility that shedding of membrane protein, but not TNFR1-Fc, binds a portion of the expressed LIGHT could be a mechanism of inactivation. LIGHT ⌬TM suggesting that it assembles as a trimer. Together, these results demonstrate that LIGHT exists in several Typical of other TNF-related ligands, LIGHT is a type II (cy- distinct molecular forms, which are directed to distinct cellular toplasmic amino terminus) transmembrane glycoprotein. How- compartments, including the extracellular space, the membrane, ever, in the absence of a transmembrane domain, LIGHT ⌬TM no and the cytosol. The realization that LIGHT is tightly linked to longer contains the necessary stop-transfer signal for proper mem- molecules of immunologic importance, including 41BBL, CD27L, brane topology and may either be routed to the cytosol or secreted. and C3, which reside in an MHC paralog, suggests that this region To determine the cellular location of LIGHT ⌬TM, nonionic de- could play an undisclosed role in genetically linked immune pa- tergent lysates or spent supernatants from LIGHT ⌬TM-trans- thology, which may now be possible to define. fected 293T cells were analyzed by precipitation with HVEM-Fc. LIGHT ⌬TM was not detectable in the culture supernatant (Fig. Acknowledgments Downloaded from 4A, lane 3), but was precipitated from the cell extract (Fig. 4A, The assistance provided by the General Clinical Research Center of ϳ lane 7) as a single band of 28 kDa. In contrast, an N terminus- Scripps Research Institute and Randi Vita for isolation of T cells and truncated soluble form of LIGHT (LIGHTt66), replaced with the G. Valkirs and T. Mikayama for the anti-LIGHT Omniclone are gratefully signal peptide from VCAM1 to direct its secretion, was readily appreciated. detected in the supernatant (Fig. 4A, lane 1) and cell lysate (Fig. 4A, lane 4) as multiple bands, which is suggestive of glycosylation References intermediates. 1. Mauri, D. N., R. Ebner, R. I. Montgomery, K. D. Kochel, T. C. Cheung, G.-L. http://www.jimmunol.org/ Yu, S. Ruben, M. Murphy, R. J. Eisenbery, G. H. Cohen, et al. 1998. LIGHT, a The extracellular domain of LIGHT contains a single predicted new member of the TNF superfamily and lymphotoxin ␣ are ligands for herpes- site for N-linked glycosylation at amino acid position Asn102. virus entry mediator. Immunity 8:21. Thus, digestion with selected glycohydrolases should provide a 2. Tamada, K., K. Shimozaki, A. I. Chapoval, Y. Zhai, J. Su, S.-F. Chen, S.-L. Hsieh, S. Nagata, J. Ni, and L. Chen. 2000. LIGHT, a TNF-like molecule, co- diagnostic picture of whether LIGHT ⌬TM is processed as se- stimulates T cell proliferation and is required for dendritic cell-mediated alloge- creted LIGHTt66 or membrane LIGHT (Fig. 4B). Endo H cleaves neic T cell response. J. Immunol. 164:4105. 3. Yu, K. Y., B. Kwon, J. Ni, Y. Zhai, R. Ebner, and B. S. Kwon. 1999. A newly high mannose structures and some hybrid oligosaccharides char- identified member of tumor necrosis factor receptor superfamily (TR6) sup- acteristic of glycoproteins that are in progress through the secre- presses LIGHT-mediated apoptosis. J. Biol. Chem. 274:13733. tory pathway, whereas PNGase F cleaves nearly all types of N- 4. Pitti, R. M., S. A. Marsters, D. A. Lawrence, M. Roy, F. C. Kischkel, P. Dowd,

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