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The Journal of Immunology, 2000, 165: 4397-4404.
Copyright © 2000 by The American Association of Immunologists

Reciprocal Expression of the TNF Family Receptor Herpes Virus Entry Mediator and Its Ligand LIGHT on Activated T Cells: LIGHT Down-Regulates Its Own Receptor1

Yannis Morel2,*, Jean-Marc Schiano de Colella2,*, Jeremy Harrop§, Keith C. Deen§, Stephen D. Holmes, Trevor A. Wattam, Sanjay S. Khandekar||, Alemseged Truneh§, Raymond W. Sweet§, Jean-Albert Gastaut{dagger}, Daniel Olive*,{ddagger} and Régis T. Costello3,*,{dagger}

* Laboratoire d’Immunologie des Tumeurs, {dagger} Département d’Hématologie, Institut Paoli Calmettes, Université de la Méditerranée, Marseille, France; and {ddagger} Institut National de la Santé et de la Recherche Médicale, Unité 119, Marseille, France; and § Departments of Immunology, Microbial and Cell Culture Science, and || Protein Biochemistry SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8+ than CD4+ T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The definition of the TNF receptor (TNFR)4 superfamily is based on the presence of cysteine-rich repeats in the extracellular region (1, 2, 3). This family consists of 19 different membrane proteins and several open viral reading frames encoding related molecules (1, 2, 3). These proteins are involved in the regulation of cell proliferation, differentiation, and death and play a central role in development of the immune response and in tumor cell killing (CD40/CD40L or Fas/FasL systems, for example). Herpes virus entry mediator (HVEM; TR2) is a recently described member of this superfamily that is broadly expressed on cells of the immune system (4, 5, 6). This molecule was first identified (4) by its ability to mediate HSV infection (HVEM) and by screening an expressed sequence tag cDNA database for sequence homology with cysteine-rich motifs of the TNFR superfamily (TR2) (5, 6). The HVEM gene maps to 1p36, close to the TNFR members CD30 (7), 4-1BB (8, 9), OX-40 (10), and TNFR-II (11). It encodes a 283-aa type I transmembrane protein containing a 50-residue cytoplasmic region that lacks a death domain, but, like other members of the TNFR superfamily, binds to a number of the TNFR- associated factor family of signal adaptors and activates transcription factors NF-{kappa}B and AP1 (12, 13). The normal cellular distribution of HVEM has mainly been determined by mRNA analysis (5, 6), showing expression in lung, spleen, thymus, monocytes, B lymphocytes, and T lymphocytes, but not in brain, liver, and skeletal muscle. More recently, HVEM protein was shown to be widely distributed on peripheral blood T and B lymphocytes, NK cells, and monocytes by flow cytometric analysis (14). By RNA analysis most solid tumor cell lines do not express HVEM, but expression is observed in hemopoietic cell lines, in particular for the myelomonocytic lineage, which is further up-regulated by phorbol esters (5). Three ligands have been identified for HVEM: the HSV surface envelope glycoprotein gD (4) and two members of the TNF family, lymphotoxin {alpha} (LT{alpha}3) and the newly described member LIGHT (TL4), which is produced by activated T cells (4, 12).

Functionally, HVEM is involved in T cell activation and can mediate a number of T cell responses, including proliferation, cytokine production, and expression of cell surface activation molecules (14). LIGHT, which also binds to the LTß receptor (15) and DcR3/TR6 (16), stimulates the proliferation of activated T cells expressing HVEM (17), stimulates NF-{kappa}B activation, and induces apoptosis in cells expressing both HVEM and LTß receptor (15) (17). LT{alpha} exists both as a secreted homotrimer (18) and as a surface heterotrimer when associated with LTß (19, 20). The secreted homotrimer binds to HVEM as well as to TNFR-I (21, 22) and TNFR-II (23).

These results indicate that HVEM plays an important role in the regulation of the immune response. In this study, we studied the expression and the mechanisms of regulation of HVEM (TR2) and LIGHT (TL4) in T lymphocytes, which are pivotal cells in the development of both the cellular and humoral immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood samples and cell separation

PBMCs from healthy donors were isolated on Ficoll-Hypaque gradients (24). T lymphocytes were isolated as the CD2-positive PBMC population, corresponding to cells that adhere to sheep erythrocytes (25) in the E-rosetting technique, but fail to adhere to plastic dishes after overnight incubation in medium and 30% FCS.

For RT-PCR analysis, positive CD4+ and CD8+ T cell isolation was performed by flow cytometry using fluorescent anti-CD4 or anti-CD8 mAbs on FACSVantage cell sorter (Becton Dickinson, Mountain View, CA). The purity of the sorted CD8+ and CD4+ cells, evaluated by reanalysis, was >=99%.

For flow cytometric experiments, we isolated CD4+ and CD8+ T cell by two rounds of negative selection using magnetic beads (Beckman Coulter, Paris, France) coated with anti-CD4 (13B8.2, D. Olive) or anti-CD8 (8E17, D. Olive) mAbs. Purity of the CD8+ and CD4+ cells by flow cytometry analysis was >=95%.

Culture conditions and dendritic cell (DC) generation

Culture experiments were performed in RPMI 1640 (Bioproducts, Walkersville, MD) with 10% FBS (Bioproducts). T lymphocytes were cultured at 106/ml. For DC generation, PBMCs were depleted of nonadherent cells by 4-h adhesion on plastic dishes. Adherent cells were then cultured in 10% RPMI 1640 (Bioproducts) with GM-CSF (Sandoz, Copenhagen, Denmark) at 100 ng/ml and IL-4 (Genzyme, Cambridge, MA) at 10 ng/ml for 6 days. The medium was replenished with cytokines every 3 days. On day 6 final maturation was induced by the addition of 50 ng/ml TNF-{alpha} (PromoCell, Heidelberg, Germany) for an additional 72 h.

T cell stimulation

For superantigen stimulation, T lymphocytes were incubated with mature autologous DCs at a 10:1 ratio, in the presence of staphylococcal enterotoxins A (SEA; Toxin Technology, Sarasota, FL) and E (SEE; Toxin Technology) at 10 ng/ml. The other stimuli used were 1 ng/ml PMA (Sigma, St. Louis, MO) and 1 µg/ml ionomycin (Sigma). In some experiments, the neutralizing LIGHT mAb 2C8 or recombinant HVEM-Fc was added at 5 µg/ml at the start of the culture.

Flow cytometric studies

For cell surface staining, cells were processed following standard procedures, and analysis was performed on a FACScan flow cytometer (Becton Dickinson). The mAbs directed against HVEM (12C5 and 20D4, both murine IgG1) and LIGHT (2C8, murine IgG2b) were generated at SmithKline Beecham by conventional hybridoma methodology from mice immunized with the respective recombinant proteins and screening the hybridomas by ELISAs. The mAbs for CD1a, CD3, CD4, CD8, CD14, CD19, CD25, CD56, CD69, and CD83 were obtained from Beckman Coulter (Hialeah, FL). The mAb for CD80 was purchased from Becton Dickinson, and the mAbs for CD86, CD40L, and FasL were obtained from PharMingen (San Diego, CA).

For intracellular detection, cells were washed twice in 1x PBS, 0.5% (w/v) BSA (Sigma), and 0.1% (w/v) saponin (Sigma). Then, 5 x 105 cells in 100 µl were stained in the same medium with the relevant mAbs. Cells were washed once in the same medium, once in PBS-BSA and then fixed for 10 min at room temperature in 1x PBS and 0.5% formaldehyde (Sigma). All data are presented after subtraction of the background represented by corresponding isotypic control mAbs.

Confocal immunofluorescence analysis

Cells were deposed on coverslips at a concentration of 1 x 106/ml and fixed in 3% paraformaldehyde (Fluka, St. Quentin Fallavier, France). Then cells were indirectly stained with biotinylated anti-HVEM 12C5 (SmithKline Beecham) or anti-LIGHT (SmithKline Beecham) mAbs, followed by streptavidin-Alaxa 488 (Molecular Probes, Eugene, OR). For membrane staining, we preincubated cells for 2 min with the red fluorescence CellTracker CM-Dil (Molecular Probes). Serial optical sections were obtained using the TCS 4D laser scanning confocal microscope (Leica, Heidelberg, Germany). Microscope settings were adjusted in to black level values when cells were stained with the mouse isotypic Ig control.

Protein synthesis inhibitor and matrix metalloprotease (MMP) inhibitors

Inhibitors were added at the beginning of the culture. The protein synthesis inhibitor cycloheximide (CHX; Sigma) was used at 10 µg/ml. The MMP inhibitor KB8301 (PharMingen), which was shown to block FasL cleavage (26, 27) was used at 1 µM according to the manufacturer’s instructions. The BB94 broad spectrum MMP inhibitor (27) (a gift from B. Mang, Institut National de la Santé et de la Recherche Médicale, Nice, France) was used at 1 µM. Cell viability was assessed by trypan blue exclusion and by propidium iodide staining for flow cytometry analysis.

Nucleic acid preparation, RT-PCR amplification, and semiquantitative analysis

Total RNA was isolated from 1.5 to 5 x 106 cells for each sample by suspension in Trizol (Life Technologies, Grand Island, NY) and extraction by phenol-chloroform, as recommended by the manufacturer. Total RNA (2.5 µg) was reverse transcribed using Moloney murine leukemia virus Superscript reverse transcriptase and random hexamers according to the manufacturer’s instructions (Life Technologies). For PCR, 2.5 µl of this cDNA was used as the target in a total volume of 25 µl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.5), 200 µM each of dNTP, 1 pmol/µl of primers, and 1.25 U of Taq polymerase (Perkin-Elmer, Norwalk, CT). The amplification was performed in a Touchdown Temperature Cycling System thermal cycler (Hybaid, Teddington, U.K.); the first cycle was at 94°C for 3 min, then annealing at 65°C for 30 s, extension at 72°C for 30 s, and denaturation at 94°C for 30 s (22 cycles for ß-actin, from 25–35 cycles for HVEM and LIGHT), terminating with 10 min at 72°C. Evaluation of the transcripts was performed by gel analysis using the Bio-Imaging Analyzer MacBAS V2.5 (Fuji Photo Film, Koshin Graphic Systems, Tokyo, Japan). Local background is subtracted for each signal. Results are expressed in arbitrary units (A.U.) as the ratio of signal intensity by ß-actin signal intensity.

Primers

The housekeeping gene ß-actin was used as a control to assess RT and PCR efficiency using the following primers: sense, 5'-ggc atc gtg atg gac tcc g-3'; and antisense, 5'-gct gga agg tgg aca gcg a-3'. The primer set for detection of HVEM was: sense, 5'-gtt cat cct gct agc tgg gtt cc-3'; and antisense, 5'-act tgg tct ggt gct gac att cct-3'. The primer set for LIGHT was: sense, 5'-gag cga agg tct cac gag gtc a-3'; and antisense, 5'-cca ggc gtt cat cca gca ca-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of HVEM and LIGHT expression in purified T lymphocytes

We first tested the regulation of HVEM and LIGHT surface expression during T cell stimulation in vitro using the superantigens SEA plus SEE, which have specificity for Vß8 and Vß5, respectively. As shown in Fig. 1Go, we observed a down-regulation of HVEM in the activated (CD25+) T lymphocyte subpopulation (Fig. 1GoC) compared with unstimulated (CD25-) T lymphocytes (Fig. 1GoA). At the same time, we observed an up-regulation of LIGHT in the stimulated T cells (Fig. 1GoD) compared with unstimulated T lymphocytes (Fig. 1GoB).



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  		FIGURE 1. Regulation of HVEM and LIGHT expression by the SEE and SEA superantigens. Purified T lymphocytes were incubated with SEE and SEA in the presence of autologous mature DCs. After 24 h of stimulation, the expression of HVEM and LIGHT was assessed by flow cytometry using anti-HVEM mAb 12C5 and anti-LIGHT mAb 2C8 together with expression of the activation marker CD25/IL-2R{alpha}. Histograms were obtained by gating on the resting T cell population (CD25-) or on the activated lymphocytes (CD25+). White curves correspond to the negative control (isotypic-matched Ab), and black curves to the HVEM or LIGHT staining. These data correspond to one representative experiment of three performed.

 
To gain insight into this apparent reciprocal regulation of HVEM and LIGHT, we evaluated their expression in several in vitro activation systems. CD28 mAbs, CD2 mAbs, PHA, IL-2, ionomycin, or PMA were tested alone or in combination. The most potent regulation of HVEM and LIGHT was observed using ionomycin along with PMA, shown in Fig. 2Go. At the beginning of culture, HVEM was expressed on all T cells, and in medium alone it remained at a constant level from time 0 through day 8 (Fig. 2GoA). In contrast, LIGHT was not detected in freshly isolated cells, and incubation with culture medium alone did not induce its expression (Fig. 2GoC). Costimulation with PMA and ionomycin induced the loss of HVEM from days 2–5, followed by a return to its original level (Fig. 2GoB). In contrast, PMA and ionomycin induced a strong expression of LIGHT that peaked on day 2 and then returned to an undetectable level on day 5 (Fig. 2GoD). A similar effect was observed with the PHA and IL-2 stimulation, although the extent of change in HVEM and LIGHT expression was less (data not shown).



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  		FIGURE 2. Flow cytometry kinetic study of HVEM and LIGHT expression by purified T lymphocytes. Purified T lymphocytes were incubated either with medium alone or with PMA/ionomycin. The expression of HVEM and LIGHT was assessed by flow cytometry using mAbs at different times until 8 days. The results are expressed as the percentage of positive cells after subtraction of the background, represented by the isotypic control. The data are presented as the mean of seven independent experiments ± SD.

 
Several studies were performed to rule out that the down-regulation of HVEM was not an artifact resulting from blockade of the receptor Ab by binding of the ligands LIGHT or LT{alpha}3. Addition of LT{alpha}3, soluble LIGHT, or PMA/ionomycin-activated T lymphocyte supernatants did not mask HVEM. Similar results were obtained with both HVEM mAbs, which have different epitope specificities. The 12C5 mAb inhibits the binding of LIGHT to HVEM, whereas the 20D4 mAb does not inhibit this interaction (R. Costello, Y. Morel, and D. Olive, unpublished observations). Finally, flow cytometric experiments using acid washing to eliminate noncovalent interactions failed to restore the detection of HVEM (data not shown). We thus conclude that the observed decrease in HVEM is the result of receptor down-modulation.

HVEM- and LIGHT-specific RNA regulation and cellular localization

To examine the mechanism by which HVEM and LIGHT protein expression is regulated, we first measured the levels of specific transcripts using semiquantitative RT-PCR. The mitogenic stimulus PMA/ionomycin induced a delayed increase in the expression of HVEM mRNA beginning around day 3 compared with the level at time zero or for incubation in medium alone (Fig. 3Go, top row). This timing precedes the reappearance of HVEM protein on the cell surface, as described above. In contrast, a rapid increase in LIGHT mRNA was observed following PMA/ionomycin stimulation (Fig. 3Go, second row), coincident with the increased cell surface expression of the LIGHT protein.



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  		FIGURE 3. RT-PCR analysis of HVEM and LIGHT transcripts in T lymphocytes. We performed semiquantitative RT-PCR on a whole population of T lymphocytes stimulated by PMA/ionomycin. We used variable numbers of cycles to compare specific signals to control ß-actin in nonsaturating conditions. Only the results corresponding to 27 PCR cycles are shown, but parallel experiments using 30 and 33 cycles were performed (data not shown). The choice of the number of PCR cycles was made to show high visibility, but nonsaturating, signals. The results presented here correspond to one representative experiment from three performed, using different healthy blood donor samples.

 
We then determined the cellular content and localization of HVEM and LIGHT during T lymphocyte activation using confocal microscopy. As shown in Fig. 4GoA, HVEM was initially distributed around the whole cell, in accordance with flow cytometric data. After a 48-h activation with PMA/ionomycin, HVEM expression was decreased but was still detected, with a "capping" phenomenon (Fig. 4GoB). The baseline expression of LIGHT protein was low, and it was localized in the cytoplasm rather than at the cell membrane (Fig. 4GoC), in agreement with the flow cytometry data. After PMA/ionomycin activation, a marked increase in LIGHT expression was observed both at the membrane and in the cytoplasm (Fig. 4GoD).



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  		FIGURE 4. HVEM and LIGHT cellular localization in T lymphocytes by confocal microscopy. Resting (A) or PMA/ionomycin-activated (B) T lymphocytes were plated on coverslips and stained with the membrane marker CellTracker CM-Dil (red fluorescence) and anti-HVEM 12C5 (green fluorescence). Resting (C) or PMA/ionomycin-activated (D) T lymphocytes were plated on coverslips and stained with the membrane marker CellTracker CM-Dil (red fluorescence) and anti-LIGHT 2C8 (green fluorescence). Serial optical sections were obtained using the TCS 4D laser scanning confocal microscope (Leica). Microscope settings were adjusted to black level values when cells were stained with the mouse isotypic Ig control.

 
Regulation of HVEM and LIGHT in the CD4+ and CD8+ subpopulations

We then examined the differential regulation of HVEM and LIGHT in CD4+ and CD8+ T lymphocyte subsets. As shown in Fig. 5Go, PMA/ionomycin induced a more potent decrease in HVEM expression in the CD8+ subpopulation compared with that in CD4+ cells, in terms of both the fraction of expressing cells (data not shown) and the mean fluorescence intensity (MFI; Fig. 5GoA). Similarly, the reciprocal up-regulation of LIGHT was more pronounced in the CD8+ subset than in CD4+ cells, particularly for the level of expression (MFI; Fig. 5GoB).



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  		FIGURE 5. Flow cytometric and RT-PCR analysis of HVEM and LIGHT expression by purified CD4+ and CD8+ T lymphocyte subsets. Purified T lymphocytes were further separated in CD4+ (X) and CD8+ ({blacksquare}) subsets by two rounds of negative selection with magnetic beads as described in Materials and Methods. The purity of the preparation, assessed by flow cytometric analysis of separated cells, was >95% in all experiments. Purified CD4+ and CD8+ T lymphocytes were separately incubated with PMA/ionomycin, and the expression of HVEM and LIGHT was assessed by flow cytometry from the beginning of the culture to day 6. Data correspond to the MFI of HVEM expression (A) and LIGHT (B) after subtraction of the background, corresponding to the isotypic control. These data correspond to one representative experiment from three performed with different healthy blood donors. We performed semiquantitative RT-PCR in the highly purified CD4+ and CD8+ subpopulations using variable numbers of cycles to compare specific signals to control ß-actin in nonsaturating conditions (C). The results shown correspond to 30 cycles, while parallel experiments using 33 and 36 cycles were also performed (data not shown). The choice of the number of PCR cycles was made to show high visibility, but nonsaturating, signals. The results presented here correspond to one representative experiment from three performed, using different healthy blood donor samples.

 
Following the results from our studies with the total T cell population, we performed RT-PCR analysis of LIGHT mRNA using highly purified (>=99%) CD4+ or CD8+ T lymphocytes. As shown in Fig. 5GoC, a higher amount of LIGHT mRNA was observed in the CD8+-activated subpopulation than in CD4+ cells. Based on normalization with the ß-actin signal, the amount of LIGHT mRNA for CD8+ increased to 3.6 A.U. upon activation, while it was 0.6 A.U. in the unstimulated condition. The amount of LIGHT mRNA was 1 A.U. for the activated CD4+ T cells, whereas it was 0.5 A.U. in the unstimulated condition.

The results of the studies reported in this and the previous section indicate that the down-modulation of HVEM is not regulated at the mRNA level, whereas both protein redistribution and mRNA levels may contribute to the up-regulation of LIGHT.

LIGHT, but not HVEM, modulation is partially mediated by metalloprotease

MMPs have been implicated in the cleavage, release, and function of several members of the TNF/TNFR family, including FasL (27), CD40L and TNF-{alpha} (28), RANKL (29), and TNFR-I/II (30). We thus tested the effects of two MMP inhibitors on the modulation of HVEM and LIGHT at the surface of activated T cells, using FasL and CD40L as positive controls. BB94 is a broad spectrum protease inhibitor, whereas MMP inhibitor (MMPI) is more selective for mitogen-activated protein kinases. The expression of both FasL and CD40L was increased by both protease inhibitors; BB94 had a greater effect Fig. 6GoB) than MMPI (Fig. 6GoA). As a negative control, the surface expression of CD25/IL-2R{alpha} was not affected by MMPI (Fig. 6GoA) or BB94 (Fig. 6GoB). Both inhibitors increased the expression of LIGHT (Fig. 6Go), and, as observed for FasL and CD40L, BB94 had the greatest effect. This protease effect on LIGHT surface expression is consistent with the detection of soluble LIGHT protein by immunoprecipitation from the supernatant of radiolabeled CD4+ T lymphocytes activated with PHA/PMA, but not from resting cells (not shown). Neither inhibitor affected the expression of HVEM on either unstimulated or PMA/ionomycin-stimulated T lymphocytes.



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  		FIGURE 6. Flow cytometric analysis of the effects of MMP inhibitors on LIGHT expression. We evaluated the effects of the MMPI (A) and BB94 (B) MMP inhibitors on the expression of LIGHT ({circ}) and HVEM (•) compared with the TNF family members FasL ({diamondsuit}) and CD40L ({blacksquare}). CD25/IL-2R{alpha} (x) corresponds to the negative control. The data are represented as: MFI induction ratio = (PMA/ionomycin + inhibitor)/(PMA/ionomycin). These data correspond to one representative experiment of three performed, using samples from different healthy blood donors. In addition, incubation with the inhibitors alone in the absence of stimulation did not modify the expression of the different surface markers compared with medium alone. Only viable cells determined by propidium iodine exclusion were analyzed.

 
Up-regulation of cell surface LIGHT is partially independent of protein synthesis

The dependence of the cell surface regulation of HVEM and LIGHT on protein synthesis was tested with the inhibitor CHX. Incubation of T lymphocytes with CHX did not modify either baseline or PMA/ionomycin HVEM expression (data not shown). In contrast, the up-regulation of LIGHT expression following PMA/ionomycin stimulation was reduced to a great extent, but not completely, by CHX preincubation (Fig. 7GoB). For comparison, the up-regulation of CD40L was almost completely inhibited by CHX preincubation (Fig. 7GoA). As controls, the expression of CD25/IL-2R{alpha}, which is induced upon T cell stimulation, was completely inhibited by CHX (Fig. 7GoC), while the expression of the early activation marker CD69 was less affected (Fig. 7GoD).



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  		FIGURE 7. Effects of the protein synthesis inhibitor CHX on LIGHT expression and CD40L in comparison with activation markers. T lymphocyte flow cytometric analysis was performed from baseline to 96 h with different markers. Cells were incubated with medium alone (x), PMA/ionomycin ({blacksquare}), or PMA/ionomycin following a 1-h preincubation with CHX ({circ}). The results are presented here with the MFI, but comparable data were obtained for the percentage of positive cells (data not shown). These graphs represent one representative experiment of three performed with samples from different healthy blood donors. Only viable cells determined by propidium iodine exclusion were analyzed.

 
The incomplete blockade of LIGHT up-regulation by CHX suggests that the increase in surface expression results in part from the relocalization of preformed molecules in the cytoplasm in addition to de novo protein synthesis. In agreement with this hypothesis and in line with the confocal microscopy results (Fig. 4Go), we observed expression of intracellular LIGHT by flow cytometry in resting T lymphocytes (Fig. 8Go). Moreover, the levels of intracellular LIGHT were greater in the CD8+ subpopulation (Fig. 8Go, lower panel) than in the CD4+ subpopulation (Fig. 8Go, upper panel), suggesting that this differential baseline content contributes to the differences in membrane expression observed between these subsets.



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  		FIGURE 8. Flow cytometric analysis of intracellular LIGHT. Purified T lymphocytes, corresponding to either the whole population (left) or purified CD4+ or CD8+ cells (right), were permeabilized and stained with anti-LIGHT mAb. White curves correspond to the negative control (isotype-matched Ig), and black curves correspond to specific staining for LIGHT. As control, we performed extracellular staining, and we failed to detect LIGHT at the cell surface of resting T lymphocytes. These data correspond to one representative experiment of three performed with different blood donors.

 
LIGHT ligation causes HVEM down-regulation

Having observed a reciprocal regulation of HVEM and one of its ligands, we tested whether engagement of HVEM by either of its TNF family ligands was responsible for its down-regulation on activated T cells. Down-modulation of HVEM was not induced by the addition of recombinant LT{alpha}, and it was not reduced by the addition of an inhibitory LT{alpha} mAb (data not shown). In contrast, incubation with the neutralizing LIGHT mAb 2C8 or with recombinant HVEM-Fc inhibited to a great extent the down-regulation of HVEM (Fig. 9Go). Thus, the induced expression of LIGHT on the cell surface or in soluble form contributes by its interaction to the down-modulation of its receptor HVEM.



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  		FIGURE 9. Effects of anti-LIGHT mAb and recombinant fusion protein HVEM-Fc on HVEM regulation. A, Purified T lymphocytes were incubated with medium alone ({diamondsuit}); PMA plus ionomycin ({blacksquare}); PMA, ionomycin, and anti-HLA DQ ({blacktriangleup}); or PMA, ionomycin, and anti-LIGHT (5 µg/ml; x). B, Purified T lymphocytes were incubated with medium alone ({diamondsuit}); PMA plus ionomycin ({blacksquare}); PMA, ionomycin, and human IgG ({blacktriangleup}); or PMA, ionomycin, and HVEM-Fc (5 µg/ml; x). Expression of HVEM was assessed at different times by flow cytometry. The anti-DQ mAb and human polyclonal Ig were used as negative controls for anti-LIGHT mAb and HVEM-Fc, respectively. These data are from one representative experiment of three performed with lymphocytes from different blood donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data provide insights into the mechanisms of regulation of HVEM and one of its ligands, LIGHT, in human T lymphocytes. Upon T cell activation, HVEM and LIGHT show reciprocal modulation on the cell surface. HVEM decreases from its constitutive level on resting cells, while LIGHT increases from a baseline that is undetectable. One week after stimulation both receptors return to their original levels. The analysis of HVEM mRNA suggests that transcriptional control or mRNA stability do not play a central role in its down-regulation following T cell activation. Although, a moderate decrease in the HVEM mRNA level occurred 6 h after activation for some donors, we also cannot exclude that this phenomenon contributes to cell surface HVEM down-regulation, but it does not seem to represent the main factor. The lack of effect by inhibitors of the MMPs that are involved in the enzymatic cleavage of other TNFR members indicates that down-regulation of HVEM is not due to the cleavage of its extracellular domain. These results together with observed capping by confocal microscopy suggest that down-regulation of this receptor following T cell activation could occur by internalization. Intracellular staining at 24 h failed to detect HVEM (data not shown) upon activation, suggesting either degradation or sequestration of the protein. Moreover, the delayed reappearance of HVEM following up-regulation of its mRNA on day 3 suggests that this material is newly synthesized, rather than recycled. The fact that addition of CHX to 48-h PMA/ionomycin-activated T cells completely inhibits HVEM reappearance supports this hypothesis (data not shown). In contrast, the reciprocal regulation of LIGHT involves different mechanisms. First, RT-PCR reveals a striking up-regulation of LIGHT mRNA transcript following T cell activation, which indicates regulation at the transcriptional level or possibly mRNA stabilization, which is reported for other TNF family members (31). Confocal microscopy and intracellular flow cytometry showed the presence of intracellular LIGHT in unstimulated T cells and a marked induction both in the cytoplasm and at the cell surface following stimulation. Along with only a partial inhibition of LIGHT up-regulation by the protein synthesis inhibitor CHX, these results indicate that both de novo synthesis and intracellular redistribution contribute to LIGHT up-regulation on the cell surface following T cell stimulation. The mechanism of the redistribution of preformed LIGHT remains unknown. Therefore, LIGHT sequence does not possess a retention signal based on homology with that described for CTLA4 (32). The basis, at least in part, for the reciprocal regulation of HVEM and LIGHT was revealed by the blockade of this process by inhibitors of this receptor/ligand interaction. The greatest effect was observed with a neutralizing LIGHT mAb, with a lesser effect on the HVEM expression level by the HVEM-Fc fusion protein. The lack of effect by addition of LT{alpha} or LT{alpha}-neutralizing Ab demonstrated that this other ligand for HVEM did not contribute to the receptor down-modulation. Thus, the down-regulation of HVEM in the presence of LIGHT antagonists could reflect incomplete blockade of this interaction or other mechanisms not yet apparent.

The complex regulation and interactions of HVEM and its ligands has to be compared with the other TNFR/TNF family member CD40/CD40L system, which is of crucial importance in the immune response (33), particularly in cytotoxic lymphocyte priming and anti-tumor immunity (34, 35). The CD40 molecule is not expressed on T lymphocytes, but it is expressed on other cells of the immune system, such as B cells and dendritic cells. Following T cell stimulation, its ligand CD40L is almost exclusively expressed on the CD4+ T lymphocytes. LIGHT is present at the cell surface of all T lymphocytes after activation, but is preferentially expressed on the CD8+ subset. In a whole T cell population the level of LIGHT expression raises to its maximum 1 or 2 days after CD40L and is ~3-fold lower. These different kinetics suggest different functions for these TNF family members. The other HVEM ligand, LT{alpha}3, is also present in all T cells, but, in contrast, preferentially in the CD4+ subset (36). Our preliminary data suggest a differential role of each ligand in HVEM regulation, since LIGHT, but not LT{alpha}3, contributes to HVEM down-regulation. Of note, when HVEM was studied by double staining with either anti-CD4 or anti-CD8 mAbs in a whole T cell population instead of separated CD4+ and CD8+ T lymphocytes, we failed to detect a difference in HVEM following stimulation (data not shown). This suggests a dialogue between the CD4+ and CD8+ subsets involving either soluble released LIGHT or intercellular contacts leading to a partial contribution of a paracrine down-regulation of HVEM in CD4+ T cells.

What could be the physiological relevance of this ligand-induced HVEM down-regulation? The interaction of HVEM with its ligand LIGHT plays a positive role in T cell proliferation. Since LIGHT is up-regulated following T cell activation, the simultaneous presence of both members of the ligand/receptor couple could induce an autocrine or paracrine activation loop. This positive activation loop could contribute to the clonal expansion of Ag-specific T lymphocytes. Nevertheless, we cannot exclude deleterious side effects of this mechanism. A paracrine effect could induce unadapted T cell proliferation or could lead to T lymphocyte exhaustion upon prolonged stimulation. As a consequence, down-regulation of HVEM by the binding of its ligand may contribute to turn off a putative self-sustained activation loop. The down-regulation of HVEM by LIGHT may also oriente the binding of the other ligand LT{alpha}3 to TNFR-I and TNFR-II, which are ubiquitous receptors with either proliferative or apoptotic effects.

The role of the HVEM ligands system is not completely elucidated, since we only know that this system participates in T cell activation (5) and can mediate, under particular circumstances, tumor apoptosis (17). Other functional implications will probably be rapidly discovered. Since HVEM is widely expressed in the pivotal cells of the immune system, such as B lymphocyte or dendritic cells (R. Costello, Y. Morel, and D. Olive, unpublished observations), we can hypothesize a role in T-B cell interaction or in DC physiology. This later point is of particular interest, since some recent publications have shown that the CD8+ T lymphocytes mediate a CD40-independent maturation of DCs (37).


   Acknowledgments
 
We thank the Departments of Molecular and Cell Culture Sciences, Protein Biochemistry, and Structural Biology at SmithKline Beecham Pharmaceuticals for production, purification, and characterization of the HVEM and LIGHT recombinant proteins used in this studies. We especially thank A.-M. Schmitt-Verhulst, C. Mawas, B. Gaugler, C. Cerdan, and Y. Collette for helpful advice and for critically reading this manuscript. We thank D. Isnardon for technical assistance with confocal microscopy.


   Footnotes
 
1 This work was supported in part by the Groupement Entreprise Français Lutte Cancer, the Association pour la Recherche contre le Cancer, the Ligues contre le Cancer des Bouches-du-Rhône et du Var, the Ligue contre le Cancer de Bastia, the Fédération Nationale des Centres de Lutte Contre le Cancer, the Etablissement Français des Greffes, the Fondation Contre la Leucémie, and the Institut SmithKline Beecham, Paris. Back

2 Y.M. and J.-M.S.d.C. equally contributed to this work and are listed by alphabetical order. Back

3 Address correspondence and reprint requests to Dr. Regis T. Costello, Laboratoire d’Immunologie des Tumeurs, Institut Paoli-Calmettes, 232 boulevard de Sainte Marguerite, 13009 Marseille, France. Back

4 Abbreviations used in this paper: TNFR, TNF receptor; CD40L, CD40 ligand; FasL, Fas ligand; HVEM, herpes virus entry mediator; D LIGHT: homologous lo Lymphotoxin, Inducible expression, complete with horpervirus protein D for HVEM, a receptor expressed by T lymphocytes; LT{alpha}, lymphotoxin {alpha}; DC, dendritic cell; SEA, staphylococcal enterotoxin A; SEE, staphylococcal enterotoxin E; MMP, matrix metalloprotease; MMPI, MMP inhibitor; CHX, cycloheximide; A.U., arbitrary units; MFI, mean fluorescence intensity. Back

Received for publication February 28, 2000. Accepted for publication July 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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