LIGHT

LIGHT Carl F. Ware* Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA * corresponding author tel: 619-678-4660, fax: 619-558-3595, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05013.

SUMMARY LIGHT is a relatively new member of the TNF family of membrane-anchored ligands that signals via two receptors: herpesvirus entry mediator (HVEM) and lymphotoxin (LT) receptor (LT R). LIGHT also interacts with the decoy receptor type 3 (DcR3) (Yu et al., 1999). LIGHT is closely related to LT and LT as defined by amino acid sequence homologies and shared receptor specificity. LIGHT exhibits inducible but transient expression on the surface of activated T lymphocytes. The receptor-binding specificity for the LT R suggests that LIGHT will be involved in lymphoid tissue organization. The high level of expression of HVEM on lymphoid cells further suggests that LIGHT may regulate T cell and B cell differentiation. LIGHT also acts as an interference factor that can block herpes simplex virus 1 (HSV) entry into cells, and may thus act as a host deterrent to virus infection.

BACKGROUND

Discovery LIGHT was discovered as a membrane protein of 30 kDa expressed on the surface of an activated T cell hybridoma (Mauri et al., 1998). This protein displayed unique receptor binding, antigenic and biophysical properties that distinguished it from all other known TNF-related proteins. These were defined through the specific binding of fusion proteins made with the ectodomain of receptors and the Fc region of IgG (Crowe et al., 1994). HVEM:Fc and LT R:Fc both specifically bound to activated T cells and precipitated a 30 kDa protein that was antigenically distinct from both LT and LT . Based on

these unique properties, LIGHT cDNA was identified from several candidate cDNAs selected from an expressed sequence tag (EST) database encoding proteins with homology to the TNF family. The acronym LIGHT refers to the ligand's features, including homology with lymphotoxins, inducible expression, competitive blockade of HSV glycoprotein D binding to HVEM, and a receptor expressed on T and B lymphocytes.

Alternative names LIGHT appears to be the generally accepted name. For the purposes of cataloging the large number of human genes, members of the TNF ligand and receptor superfamilies have been assigned numerical indicators (www.gene.ucl.ac.uk/nomenclature/). LIGHT is designated as TNFSF-14 by this system. LIGHT has also been referred to as HVEM ligand (Harrop et al., 1998) but this overlooks homology with lymphotoxins.

Structure LIGHT is a type 2 (N-terminal cytosolic tail) transmembrane glycoprotein of 240 amino acids. Sequence homology to the TNF family implies that LIGHT will have a primary conformation as an antiparallel sandwich structure that assembles into a homotrimer. Species conservation between human and mouse LIGHT is 76%.

Main activities and pathophysiological roles Little is known about the normal cellular responses induced by LIGHT via interaction with HVEM.

514 Carl F. Ware Studies by Tan (Tan et al., 1997) suggests that HVEM may play a role as a costimulatory receptor for lymphocytes. Anti-HVEM blocks the secretion of several cytokines by activated T cells, such as IFN and IL-2. LIGHT produced as a soluble recombinant protein can induce IFN secretion by peripheral blood lymphocytes (Zhai et al., 1998). LIGHT also triggers cell death/growth inhibition of the HT29 adenocarcinoma (Harrop et al., 1998; Zhai et al., 1998). The signaling of both HVEM and LT R have been postulated to mediate cell death (Zhai et al., 1998). Tissue culture models demonstrate that activation of the LT R can induce cell death in the HT29 adenocarcinoma cell line and cause tumor regression when transplanted into mice (Browning et al., 1996). Studies by Zhai et al. (1998) using MDA-MB-231 tumor cells transfected with LIGHT cDNA have shown the growth suppression of tumor xenografts, with a neutrophil infiltration and the necrosis of LIGHT-expressing tumor cells. Like LT1 2, LIGHT may have an antitumor function in certain contexts in vivo. Biologic roles for LIGHT have yet to be fully revealed. However, the receptor-binding properties of LIGHT imply likely roles in lymphoid tissue structure and function. LT R plays a major role in the formation of secondary lymphoid tissue during embryogenesis (Futterer et al., 1998) and regulates the microarchitecture of the spleen, primarily in the segregation of T and B cell zones and the formation of the follicular dendritic cell network in germinal centers. LT or LT knockout mice (De Togni et al., 1994; Banks et al., 1995; Koni et al., 1997; Alimzhanov et al., 1997) present with lymphoid tissue phenotypes similar but not identical to those of the LT Rÿ/ÿ mice. This indicates that LIGHT is not redundant with LT or LT for these functions. However, differences between the LT/ ligand and LT R knockouts, or partial phenotypic differences observed between LTÿ/ÿ and LT ÿ/ÿ, may identify candidate traits mediated by LIGHT. In this regard, Pfeffer's analysis of the LT R knockout suggests that LIGHT may play a partially redundant role with LT1 2 in the affinity maturation of the antihapten Ig response (Futterer et al., 1998). LIGHT could also participate in the formation of splenic B cells clusters, as surmised by aberrant PNA‡ cell clusters around the central arteriole in LT Rdeficient mice, a unique phenotype for LT Rÿ/ÿ mice (Futterer et al., 1998). LIGHT interferes with the entry of herpes simplex virus 1 (HSV-1) into CHO cells transfected with HVEM, suggesting that LIGHT can act as an antiviral deterrent (Mauri et al., 1998). HSV envelope

gD also binds HVEM and causes a similar interference phenomenon (Johnson and Spear, 1989). Thus interference with virus infectors probably results from the ligand-mediated downmodulation of HVEM. Envelope glycoprotein D competes with HVEM, but not LT R, binding to membraneanchored LIGHT. This observation suggests that HSV may be able to modify LIGHT±HVEM signaling pathways and subsequent cellular responses. Of the several entry routes that HSV can utilize (Geraghty et al., 1998), HVEM may serve as a major entry route for T lymphocytes (Montgomery et al., 1996). Interestingly, HSV-infected fibroblasts rapidly inactivate the cytolytic capacity following cell-to-cell contact of CTL or NK cells (Posavad and Rosenthal, 1992), suggesting that HSV may use the HVEM entry route as a mechanism of immune suppression.

GENE AND GENE REGULATION

Accession numbers GenBank: Human LIGHT cDNAs: AF036581, AF064090

Sequence These two cDNA sequences differ slightly in the coding region, with a G to A change resulting in a nonconservative substitution at lysine 214 to glutamic acid. This substitution is located in a highly conserved region in the G strand. Both forms appear to interact with HVEM (Mauri et al., 1998; Harrop et al., 1998) and therefore represent an allelic difference.

Chromosome location LIGHT is located on human chromosome 16p11.2 as determined by FISH (Zhai et al., 1998). Mouse chromosome 7D-E1.1.

Cells and tissues that express the gene See Table 1. A comprehensive examination of all cell types that produce LIGHT has not been reported. However, LIGHT expression by T cells requires activation stimuli similar to those for the related ligands LT and LT . LIGHT is produced by phorbol ester and ionomycin-activated II-23.D7 cells,

LIGHT Table 1

515

Expression patterns of LIGHT mRNA

Cell lines/tissues

Detected by

Lymphocytic cell lines II-23 CD4‡ T cell hybridoma

RT-PCR (Zhai et al., 1998)

TIL1200 T cell

RT-PCR (Zhai et al., 1998)

TIL1235 T cell

RT-PCR (Zhai et al., 1998)

THP-1 monocytic line

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Nonlymphoid cell lines MCF10A breast epithelial transformed

RT-PCR (Zhai et al., 1998)

Lymphoid tissues PBL (activated anti-CD3)

RT-PCR (Zhai et al., 1998)

CD4‡ T cells

RT-PCR (Zhai et al., 1998)

Granulocytes

RT-PCR (Zhai et al., 1998)

Monocytes

RT-PCR (Zhai et al., 1998)

Spleen

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Lymph nodes

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

PBL

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Thymus

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Other tissues Appendix

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Brain

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Placenta

Northern blot analysis (Harrop et al., 1998; Mauri et al., 1998)

Heart, kidney, liver, lung, colon (weak)

a CD4‡ T cell hybridoma, but in contrast to LT or LT , LIGHT is also expressed by the monocytic cell line THP-1 following treatment with phorbol ester. In one case, LIGHT mRNA was detected in the transformed MCF10A breast epithelial line (Zhai et al., 1998). These results suggest that LIGHT may have a broader tissue expression pattern than LT and LT , which is restricted to activated T and B lymphocytes and NK cells. Mitogen-activated CD4‡ and CD8‡ T cell subsets from peripheral blood have readily detectable LIGHT mRNA. LIGHT was also detected in granulocytes and monocytes by RT-PCR (Zhai et al., 1998). By northern blot analysis, LIGHT mRNA is expressed abundantly in spleen and lymph

nodes, less so in peripheral blood, thymus, and appendix, and weakly in bone marrow. Visceral organs, heart, colon, small intestine, lung, and liver exhibit weak expression. Reports of expression in the brain vary (Harrop et al., 1998; Mauri et al., 1998).

PROTEIN

Accession numbers GenBank: LIGHT: AF036581, AF064090

516 Carl F. Ware

Sequence See Figure 1.

Description of protein The primary structure of LIGHT that is predicted from the cDNA sequence contains 240 amino acids, no predicted signal cleavage site, and a hydrophobic region characteristic of a type 2 transmembrane protein (Figure 1a). LIGHT contains an N-terminal cytosolic domain of 37 residues that precedes a stretch of 22 hydrophobic residues. The extracellular domain consists of a short membrane extension of 39 residues prior to the receptor-binding domain. The predicted molecular weight is 26,350; however, when LIGHT cDNA is expressed by HEK 293 cells, the observed molecular mass is 28±29 kDa and the natural protein from activated II-23 T cell line is 30 kDa (Table 2). The analysis of a truncated form of LIGHT (position G85) reveals a soluble protein that retains receptor binding activity, and analytical ultracentrifugation has shown a solution mass of 64 kDa consistent with the formation of a stable homotrimer (Harrop et al., 1998).

Discussion of crystal structure LIGHT has not been crystallized. The significant homology of LIGHT to LT, TNF, and CD40L, all of which have been crystallized, allows the construction of a theoretical three-dimensional model (Guex and Peitsch, 1997) (Figure 2).

Important homologies LIGHT exhibits significant sequence homology with the C-terminal receptor-binding domains of LT

(34% identity), Fas ligand (31%), 4-1BB ligand (29%), TRAIL (28%), LT (27%), TNF (27%), and CD40L (26%) (Figure 1). The ligands for CD30, CD27, and Ox40 exhibit the weakest homology (12±18% identity, not shown in this alignment). No sequence homology is found with HSV-1 envelope gD. Sequence homology is primarily limited to the residues forming the strand scaffold, suggesting that LIGHT, like LT, CD40L, and TNF, folds into an antiparallel sandwich structure and assembles as a trimer. The similarity of LIGHT to lymphotoxins outside the scaffold regions is seen in the conservation of tyrosine 173 located in the D±E loop, a contact region in LT for TNFR60 (Banner et al., 1993).

Posttranslational modifications LIGHT contains a single N-linked glycosylation site (Asn102) that lies within the major receptor-binding loop (A±A0 strand). Activated T cells do not appear to produce a soluble form of LIGHT. One report suggests that LIGHT may be shed by the breast carcinoma line MDA-MB-231 (Zhai et al., 1998).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Activated T cells produce LIGHT. For the II-23 T cell hybridoma, both phorbol ester and ionomycin are required for the expression of LIGHT, while PMA is sufficient for LT/ expression (Mauri et al., 1998). In fact, the combination of PMA and ionomycin caused a significant decrease in the expression of the LT complex. Other agents that activate T cells,

Figure 1 Sequence of LIGHT and alignment with TNF superfamily. Amino acid sequence deduced from the cDNA sequence. The asterisk indicates the position of the predicted Nglycosylation site. Reproduced with permission from Mauri et al. (1998).

LIGHT

517

Table 2 Selected physical properties of human LIGHT, LT, and LT Propertya

LIGHT

LT

LT

Amino acids

240

171

233

Signal cleavage site

None

34

None

Cytoplasmic tail

37

None

29

Transmembrane domain

38±54

None

29±45

Membrane extension domain

39

None

36

Receptor-binding domain

147

137

160

Sequence

26

17

26

Observed

30

25

33

Homotrimer

LIGHT3

LT3

No

Heterotrimer

No

LT1 2/LT2

LT1 2

Secreted forms

No

Yes (LT3)

No

N-linked glycosylation sites

1

1

1

Molecular mass (kDa)

Quaternary structure:

1

a

Analyzed using PSORT (http://psort.nibb.ac.jp: 8800).

Figure 2 Theoretical model of LIGHT. This model was generated using Swiss-Model (www.expasy.ch/swissmod/ SWISS-MODEL.html). The model is based on sequence Ser103 to V240 and is displayed as a homotrimer.

RECEPTOR UTILIZATION LIGHT binds to HVEM (Mauri et al., 1998) and LT R (Mauri et al., 1998), and DcR3 (Yu et al., 1999).

IN VITRO ACTIVITIES

In vitro findings See Table 3.

Bioassays used such as phytohemagglutinin or anti-CD3, or specific antigens, induce the expression of LIGHT. As measured by the induction of LIGHT mRNA, other cell types, including monocytes and granulocytes, may express LIGHT. This has not yet been confirmed at the level of protein expression. Phorbol ester is sufficient to stimulate production by the THP-1 monocytic line (Harrop et al., 1998).

LIGHT, like LT, TNF, and LT1 2, induces apoptosis in HT29 adenocarcinoma cells in the presence of IFN (Zhai et al., 1998), as measured by MTT dye reduction or the inhibition of cell growth ([3H]thymidine incorporation) (Harrop et al., 1998). LIGHT induces IFN secretion by peripheral blood lymphocytes (Zhai et al., 1998) and exhibits weak NFB activation (Harrop et al., 1998). LIGHT can be detected on the surface of activated lymphocytes using flow cytometry with HVEM:Fc.

518 Carl F. Ware Table 3 Some in vitro response assays for LIGHT Cellular responses to LIGHT

Assay

References

Cell death/growth inhibition

HT29 adenocarcinoma with IFN

Zhai et al., 1998; Harrop et al., 1998

IFN secretion

Peripheral blood lymphocytes

Zhai et al., 1998

Interference of herpes simplex virus (HSV) infection

HVEM-dependent infection of CHO cells by HSV

Mauri et al., 1998

NFB activation

B-dependent luciferase assay

Harrop et al., 1998

LT R:Fc will also detect LIGHT, but may simultaneously detect LT1 2.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Species differences None has been reported, but see HVEM as the species differences between human and mouse HVEM are significant (Hsu et al., 1997).

Knockout mouse phenotypes These are not known, although see LT R.

Interactions with cytokine network LIGHT induces the secretion of IFN by peripheral blood lymphocytes (Zhai et al., 1998).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects No pathophysiological roles have been defined, although the ability of LIGHT to interfere with herpes simplex virus 1 (HSV-1) entry in tissue culture models suggests that this cytokine may play a role in antiviral defense mechanisms in vivo (Mauri et al., 1998).

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LIGHT proliferation of T cells and inhibits HT29 cell growth. J. Biol. Chem. 273, 27548±27556. Hsu, H., Solovyev, I., Colobero, A., Elliott, R., Kelley, M., and Boyle, W. J. (1997). ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J. Biol. Chem. 272, 13471±13474. Johnson, R. M., and Spear, P. G. (1989). Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection. J. Virol. 63, 819±827. Koni, P. A., Sacca, R., Lawton, P., Browning, J. L., Ruddle, N. H., and Flavell, R. A. (1997). Distinct roles in lymphoid organogenesis for lymphotoxins  and revealed in lymphotoxin -deficient mice. Immunity 6, 491±500. Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu, G.-L., Ruben, S., Murphy, M., Eisenbery, R. J., Cohen, G. H., Spear, P. G., and Ware, C. F. (1998). LIGHT, a new member of the TNF superfamily and lymphotoxin  are ligands for herpesvirus entry mediator. Immunity 8, 21±30. Montgomery, R. I., Warner, M. S., Lum, B., and Spear, P. G. (1996). Herpes simplex virus 1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427± 436.

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Posavad, C. M., and Rosenthal, K. L. (1992). Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity. J. Virol. 66, 6264±6272. Tan, K. B., Harrop, J., Reddy, M., Young, P., Terrett, J., Emery, J., Moore, G., and Truneh, A. (1997). Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells. Gene 204, 35±46. Yu, K. Y., Kwon, B., Ni, J., Zhai, Y., Ebner, R., and Kwon, B. S. (1999). A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J. Biol. Chem 274, 13733±13736. Zhai, Y., Guo, R., Hsu, T.-L., Yu, G.-L., Ni, J., Kwon, B. S., Jiang, G., Lu, J., Tan, J., Ugustus, M., Carter, K., Rojas, L., Zhu, F., Lincoln, C., Endress, G., Xing, L., Wang, S., Oh, K.-O., Gentz, R., Ruben, S., Lippman, M. E., Hsieh, S.-L., and Yang, D. (1998). LIGHT, a novel ligand for lymphotoxin receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer. J. Clin. Invest. 102, 1142±1151.