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* Laboratory of Transplantation Immunology and
Nephrology Service of Notre Dame Hospital, Center Hospitalier de l’Universite de Montreal, University of Montreal, Montreal, Canada; and
Human Genome Sciences, Rockville, MD
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R on the cell surface, and TR6 in solution.
In the present study, we report soluble TR6-Fc or solid-phase
TR6-Fc costimulated proliferation, lymphokine production, and
cytotoxicity of human T cells in the presence of TCR ligation. These
costimulating effects were blocked by soluble LIGHT but not by soluble
Fas-Fc. TR6-Fc could also effectively costimulate
gld/gld mouse T cells. We further demonstrated that TR6
bound to both Th1 and Th2 cells, according to flow cytometry, and that
the association was inhibited by soluble LIGHT. Cross-linking Th1 and
Th2 cells with solid-phase TR6-Fc along with a suboptimal concentration
of anti-CD3 enhanced proliferation of both Th1 and Th2 cells, and
augmented Th1 but not Th2 lymphokine production. These data suggest
that TR6 delivers costimulation through its ligand(s) on the T cell
surface, and at least the major part of such costimulation is via
LIGHT. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is entirely secreted) are type
II membrane proteins, and can exert their effect through cell-cell
contact (1). Many members, such as TNF-
(4), CD40 ligand
(CD40L)4
(4), Fas ligand (FasL) (5), and TNF-related
activation-induced cytokine (TRANCE) (6) can be
cleaved from cell surfaces. The cleaved parts of these members have
demonstrated (as in the case of TNF-) or conceivable biological
functions that involve interaction with their respective receptors.
LIGHT/TL5 is a new member of the TNF family (7),
with its protein expressed on activated T cells (7) and
immature dendritic cells (8). Cell surface LIGHT can be
cleaved by matrix metalloproteinase (9). It is a ligand
for TR2/Herpesvirus entry mediator, lymphotoxin receptor
(LTR), and DcR3/TR6, all of which are TNFR family members
(7, 10, 11). Recent studies show that LIGHT can
costimulate T cell responses via TR2, which is constitutively expressed
at both protein and mRNA levels in most lymphocyte subpopulations,
including CD4 and CD8 T cells (12, 13). LIGHT, expressed
on COS cells or anchored on a solid phase, augments T cell
proliferation as well as lymphokine production (3, 8).
Molecules that presumably interfere with the interaction between LIGHT
and TR2 can down-regulate T cell responses: soluble recombinant TR2-Fc
inhibits a three-way MLR (13) or dendritic cell-stimulated
alloresponse of T cells (8), an antagonistic mAb against
TR2 represses proliferation and lymphokine production by CD4 T cells
(12), soluble LTR-Fc suppresses solid-phase
LIGHT-augmented T cell proliferation (3), and in vivo
administration of LTR-Fc leads to amelioration of mouse
graft-vs-host disease (3). Taken together, these pieces of
evidence show that LIGHT acts on TR2 as a costimulator of T cell
activation. Moreover, LIGHT can induce apoptosis in cells expressing
both TR2 and LTR (14), although Rooney et al.
(15) reported that LTR is necessary and sufficient for
LIGHT-mediated apoptosis in tumor cells. Because LTR is not
expressed on lymphocytes (16), LIGHT is unlikely to cause
apoptosis in these cells.
TR6 is a new member of the TNFR family. Human TR6 lacks an apparent transmembrane domain in its sequence, and is a secreted protein (10, 17). In the immune system, TR6 mRNA is expressed at high levels in lymph nodes and the spleen (17, 18), while its expression in the thymus and PBLs is weak or undetectable, respectively. TR6 has three known ligands, i.e., FasL, LIGHT, and TL1A. TR6 can bind to FasL and inhibit the interaction between Fas and FasL. Consequently, FasL-induced apoptosis of lymphocytes and of several tumor cell lines can be repressed by TR6 (17). TR6 can also bind to LIGHT (10, 11). We have recently reported that human TR6-Fc can compete with TR2 for binding to LIGHT on human T cells, suppress CTL and lymphokine production in mouse lymphocytes, and inhibit mouse heart allograft rejection (10). These findings have raised the possibility that TR6 inhibits LIGHT-triggered costimulation via TR2 in T cells. The third known ligand of TR6 is TL1A, which is a new member of the TNF family, and is predominantly expressed on endothelial cells (19). TR6 can repress TL1A-augmented lymphokine secretion and the graft-vs-host response (19).
Our current study has revealed another layer of complexity in the interactions between TR2, TR6, and LIGHT. We present evidence in this study that TR6 ligand on the cell surface actually transduces costimulating signals into T cells, and enhances T cell responses to mitogens and alloantigens. At least a part of such reverse signaling was mediated by LIGHT. Thus, although a ligand, LIGHT can function as a receptor as well. The biological significance of this finding is discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant TR6-Fc, TR6, TR11-Fc, LIGHT, and FasL were prepared as described in our previous publications (10, 20).
mAbs (clones 17B07 and SK9E2) against TR6 were prepared as follows. BALB/c mice were immunized i.p. with 50 µg/100 µl of TR6 emulsified in 100 µl of CFA. Three additional s.c. injections of 25 µg of TR6 in IFA were given at 2-wk intervals. The animals were rested for a month before receiving the final i.p. boost of 25 µg of TR6 in PBS. Four days later, splenocytes from one of the immunized mice were fused with 2 x 107 P3 x 63Ag8.653 plasmacytoma cells using polyethylene glycol 1500 (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s instructions. After fusion, the cells were resuspended in 400 ml of hypoxanthine/aminopterin/thymidine medium supplemented with 20% FCS and 4% of hybridoma supplement (Roche Applied Sciences), and distributed into 96-well plates (200 µl/well). Hybridomas were screened for specific Ab production by ELISA using TR6-coated plates. Positive hybridoma supernatants were checked for Ig isotypes using mouse Iso-strip kit (Roche Applied Sciences). mAb affinity was ranked by ELISA according to an approach described earlier (21). Hybridomas producing high-affinity mAbs were cloned by limiting dilution. Cloned hybridoma cells were injected in pristine-primed BALB/c mice (3 x 106cells/mouse) for ascites production. The Abs were purified from the ascites by protein G affinity chromatography using the Acta fast protein liquid chromatography system (Amersham Pharmacia Biotech, Piscataway, NJ).
Preparation and culture of PBMCs, T cells, Th1 cells, Th2 cells, and mouse spleen cells
Adult PBMCs were prepared by Lymphoprep gradient (Nycomed, Olso, Norway), and T cells were prepared from PBMC by sheep RBC rosetting as described elsewhere (22), or by negative selection (deletion of cells positive for CD11b, CD16, CD19, CD36, and CD56) with magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The mouse mononuclear spleen cells were prepared by lysing RBCs in the total spleen cells with 0.84% NH4Cl. The cells were cultured in RPMI 1640 supplemented with 10% FCS, L-glutamine, and antibiotics. RPMI 1640, FCS, penicillin-streptomycin, and L-glutamine were purchased from Life Technologies (Burlington, Ontario, Canada). [3H]Thymidine uptake was measured as described previously (22, 23).
For Th1 and Th2 cell generation, cord blood mononuclear cells were
isolated by density gradient on Histopaque-1077 (Sigma-Aldrich, St.
Louis, MO). Monocytes from cord blood mononuclear cells were deleted by
culture dish adhesion for 1 h at 37°C, and the resulting
lymphocyte fraction was cultured with 2 µg/ml PHA (Sigma-Aldrich) in
the presence of Th1/Th2 polarizing Abs and cytokines. Th1
differentiation was triggered by addition of 5 ng/ml IL-12 and 5
µg/ml anti-IL-4; Th2 differentiation was initiated by addition of
5 ng/ml IL-4, 5 µg/ml anti-IL-12, and 5 µg/ml anti-IFN-
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After 72 h, cells were cultured in medium containing 5 ng/ml IL-2.
After an additional 11–14 days of culture, >99% of the cells were
CD3+ T cells according to flow cytometry
analysis; their Th1 and Th2 phenotype was confirmed by their lymphokine
production profile. These cells were washed once with serum-free RPMI
medium and starved in IL-2-free medium for 3 h. They were then
cultured at 1–2 x 105 cells/well in
96-well plates, which were coated with anti-TCR (3 µg/ml)
in combination with various amounts of solid-phase TR6-Fc.
Proliferation of these cells was measured by
[3H]thymidine uptake 2–3 days later. The mAbs
used in this section were from BD PharMingen (San Diego, CA), and ILs
were from R&D Systems (Minneapolis, MN).
Lymphokine assays
IL-2, IL-5, IL-6, IL-10, IFN-, TNF-, and GM-CSF in culture
supernatants were measured by commercial ELISA kits from R&D
Systems.
Flow cytometry
Th1 or Th2 cells (1 x 106) without further stimulation, or stimulated with solid-phase CD3, were stained with TR6-Fc (15 ng/sample) followed by goat F(ab')2 anti-human IgG-PE (Southern Biotechnology Associates, Birmingham, AL). In some samples, TR6 without the Fc tag, LIGHT, or anti-TR6 mAb was present as an inhibitor (5 µg/sample) during the staining process. The cells were washed and resuspended in buffer containing 0.5 µg/ml propidium iodide; propidium iodide-negative live cells were gated and analyzed by flow cytometry.
Cytotoxic T cell assay
CTL activity of 
T cells was assayed as follows. Human
PBMC (4 x 105 cells/200 µl/well) were
stimulated with mitomycin C-treated Daudi cells (0.7 x
105 cells/well) in round-bottom 96-well plates in
the presence of 10 U/ml IL-2 for 6 days. Normal human IgG (20 µg/ml),
TR6-Fc (20 µg/ml), or LIGHT (10 µg/ml) was added to the culture in
the beginning. On day 6, cells given the same treatment in the 96-well
plate were pooled and counted, and their CTL activity was measured by a
standard 4-h 51Cr-release assay using
51Cr-labeled Daudi cells (1.5 x
105 cells/well) as targets at different E:T
ratios. To test the effect of solid-phase TR6-Fc, the round-bottom
wells were precoated with TR6-Fc (0.5 µg/50 µl/well) at 4°C
overnight and washed with PBS. PBMC (4 x
105 cells/250 µl/well) and mitomycin C-treated
stimulator Daudi cells (0.7 x 105
cells/well) were cultured in these wells with IL-2 (10 U/ml) in the
absence or presence of soluble LIGHT (20 µg/ml). On day 3, 70 µl
culture supernatant/well was replaced with fresh regular medium. All
the other procedures of the CTL assay were the same as described above,
except that the 51Cr-release assay was conducted
on day 5 instead of day 6.
The lysis percentage of the test samples was calculated as follows:
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T cell CTL activity, PBMCs from donor A were used as
responder cells, and mitomycin C-treated PBMCs from donor B were used
as stimulators. PBMCs from donor B without mitomycin C treatment were
stimulated with solid-phase anti-CD3 and anti-CD28, and used as
target cells on day 6 for the 4-h 51Cr-release
assay. All the other procedures were the same as described above. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We made an intriguing discovery in the course of our study on the
role of TR6 in immune regulation: soluble TR6-Fc augmented response of
human PBMC stimulated with different concentrations of PHA (0.05, 0.1,
0.5, and 1 µg/ml) in the presence of TR6-Fc (20 µg/ml) (Fig. 1
A). TR6-Fc greatly enhanced
the PBMC proliferation when PHA was at suboptimal concentrations (0.1
and 0.5 µg/ml). Using a suboptimal concentration of PHA (0.2
µg/ml), we further demonstrated that soluble TR6-Fc could enhance
PBMC proliferation in a dose-dependent manner from 0.3–30 µg/ml
(Fig. 1B). To ensure that the effect of TR6-Fc plus PHA was
not due to a shift in proliferation kinetics compared with that of PHA
or TR6-Fc stimulation alone, PBMC stimulated with PHA (0.2 µg/ml),
TR6-Fc (10 µg/ml), or both were harvested at 48, 72, and 96 h.
The result (Fig. 1C) showed that at no time points did
TR6-Fc or suboptimal PHA alone lead to significant proliferation,
unlike PHA plus TR6-Fc, ruling out a possible kinetic shift. Soluble
TR6-Fc (10 µg/ml) also augmented suboptimal soluble anti-CD3 (100
ng/ml)-stimulated proliferation, as shown in Fig. 1D. The
results of this section demonstrate that TR6-Fc can stimulate PBMC and
T cells, likely via its cell surface ligand(s).
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E). The solid-phase TR6-Fc in this experiment
was prepared by coating the culture wells with nonneutralizing
anti-TR6 mAb (clone 17B07) followed by TR6-Fc.
[3H]Thymidine uptake was measured 72 h
later. Solid-phase TR6 enhanced T cell proliferation in the presence of
suboptimal PHA concentration, indicating that it delivers a
costimulation signal through its binding partners on T cells. TR6 has
two known ligands on T cell surface, LIGHT and FasL. Ideally, to
identify which ligand was involved in the process, soluble LIGHT and
FasL should be used as blockers in the culture. The former was used for
this purpose. However, because exogenous FasL in solution might lead to
apoptosis of activated T cells, it could not be used as a blocker for
this purpose. Thus, we used soluble Fas-Fc, which would bind to FasL
and block the interaction between FasL and TR6. The result showed that
LIGHT but not Fas-Fc inhibited the proliferation, suggesting that LIGHT
is a likely molecule through which TR6 induces activation of T
cells.
One could argue that soluble LIGHT might block the binding of TR6 to
FasL, and soluble Fas might have lower affinity than TR6 in FasL
binding (although there is no evidence for this assumption) and thus
cannot effectively compete with TR6 for binding to FasL. With such
assumptions, whether it is the LIGHT that transduced signals into the
cells remains in doubt. To gain additional evidence for LIGHT-mediated
reverse signaling, solid-phase TR6 was used to stimulate
gld/gld mouse spleen cells, which have nonfunctional FasL.
Human TR6 can effectively bind to mouse FasL (data not shown) and LIGHT
(10). gld/gld spleen cells responded well to
solid-phase TR6 stimulation (Fig. 1F), suggesting that the
major part of the reverse signaling is not via FasL. Solid-phase TR2,
which binds to LIGHT but not FasL, costimulated mouse T cell
proliferation when they were triggered by suboptimal solid-phase
anti-CD3 (Fig. 1G). Taken together, these data further
indicate that LIGHT is the main molecule mediating the TR6-triggered
reverse signaling.
To compare the efficacy of dimeric TR6-Fc vs monomeric TR6 without Fc
on T cell stimulation, and to test the hypothesis whether the observed
stimulatory effect of TR6-Fc was due to blocking of a putative negative
regulatory autocrine loop by TR6-Fc or TR6 leaked into
solution (see Discussion for further elaboration), we
coated these molecules directly on wells (1 µg/50 µl/well during
coating). PBMCs were cultured in these wells in the presence of a
suboptimal concentration of PHA (0.2 µg/ml) for 72 h, and
thymidine uptake was measured. As shown in Fig. 1H, TR6-Fc
but not TR6 could enhance T cell proliferation. This showed that the
power of cross-linking is correlated to the efficacy of TR6
stimulation. Moreover, this indicates that the existence of a negative
regulatory loop is unlikely, because if so, monomeric TR6 without Fc
leaked into solution should more effectively enhance T cell
proliferation than dimeric TR6-Fc.
TR6-Fc costimulation led to augmentation of lymphokine production by PBMC
Human PBMCs were cultured in the presence of a suboptimal
concentration of PHA (0.2 µg/ml), PHA plus TR6-Fc (20 µg/ml), or
PHA plus control recombinant protein TR11-Fc (20 µg/ml). The
cytokines secreted into the supernatants at 24, 48, and 72 h were
tested with ELISA and the results are presented in Fig. 2A. Because PHA was used at a
suboptimal concentration, it induced minimal cytokine production. When
TR6-Fc was added to the culture, it drastically induced production of
cytokines such as IL-2, IL-6, IL-10, GM-CSF, IFN-, and TNF-. In
contrast, control protein TR11-Fc in combination with PHA did not
augment cytokine production. Some Th2-type cytokines, such as IL-4 and
IL-5, were of very low levels in this system, and no changes were
detected (data not shown). Thus, costimulation from TR6-Fc led to
augmented cytokine production in PBMC.
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and IFN- production at 48 h by T cells (Fig. 2B,
top two panels), as with total PBMC. Soluble LIGHT, but not
a control protein, TR11-Fc, prevented the augmentation of lymphokine
production. This suggests that costimulation of TR6 is delivered to the
T cells through a cell surface TR6 ligand, with LIGHT being a likely
candidate. When stimulated with a suboptimal concentration of PHA in
solution and solid-phase TR6, these T cells also augmented IL-2 and
GM-CSF production, compared with PHA stimulation alone (Fig. 2B, bottom two panels). TR6 bound to LIGHT expressed on Th1 and Th2 cells
LIGHT expression is up-regulated on activated T cells
(7); we showed previously that TR6 specifically bound to
LIGHT expressed on those cells (10). In this study, we
examined LIGHT expression and association of TR6 with LIGHT on Th1 and
Th2 cells. These cells were stimulated with solid-phase anti-CD3
overnight. As shown in Fig. 3, row
1, TR6-Fc bound to anti-CD3-activated Th1 and Th2 cells
(shaded areas), but not to ones without activation (solid lines).
Unlabeled soluble TR6 (Fig. 3, row 2 without the Fc tag) and
anti-TR6-mAb (Fig. 3, bottom row) inhibited the TR6
staining, indicating that the binding was not nonspecific. Soluble
LIGHT (Fig. 3, row 3) effectively blocked the staining of
TR6-Fc. These results suggest that the ligand of TR6 (likely LIGHT) is
expressed on activated Th1 and Th2 cells.
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We next assessed whether TR6 differentially promoted Th1 or Th2
function. In the presence of suboptimal solid-phase anti-TCR,
solid-phase TR6 stimulated proliferation of both Th1 and Th2 cells
similarly and dose-dependently (Fig. 4, A and B); TR6-Fc alone without anti-TCR
signaling had no effect on these cells (data not shown). As these Th1
and Th2 cells were >99% pure T cells, this experiment excluded the
possibility that the effect of TR6 was indirect via dendritic cells or
monocytes/macrophages. TR6 neutralizing mAb (clone SK9E2) suppressed
TR6-enhanced Th1 and Th2 proliferation in a dose-dependent manner,
while control mouse IgG had no effect (Fig. 4, C and
D), indicating that the effect of TR6 is specific.
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, E and F).
As controls, anti-CD28 and suboptimal anti-TCR on the
solid phase resulted in dramatic IFN- production by Th1 but not Th2
cells (74,936 ± 56 vs 72 ± 23 pg/ml), and marked IL-5
production by Th2 but not Th1 cells (586.9 ± 16.5 vs 1.1 ±
0.14 pg/ml), confirming the Th1 and Th2 phenotype of the cells. When
solid-phase TR6-Fc was used along with anti-TCR, it
significantly increased IFN- production by Th1 cells (42,587 ±
4,535.2 pg/ml), compared with anti-TCR stimulation alone
(8,064.5 ± 223 pg/ml); this augmentation was blocked by
anti-TR6 mAb in a dose-dependent manner, showing the specificity of
the TR6 stimulation (Fig. 4E). IL-5 production by the Th1
cells was negligible (<5–10 pg/ml) with such stimulation, as
expected, since IL-5 is a Th2 lymphokine. Th2 cells stimulated with
TR6-Fc and anti-TCR produced little IFN- (<30 pg/ml), as
expected, since IFN- is a Th1 lymphokine; however, they also failed
to produce Th2 lymphokine IL-5 (Fig. 4F). The results of
this section show that TR6 costimulation preferentially augments Th1
but not Th2 cell function in terms of production of certain
lymphokines, although it stimulated similar proliferation responses of
both cell types. TR6 and LIGHT on CTL development
To further assess the functional consequence of TR6 costimulation,
we examined the CTL development of PBMC in the presence of soluble
TR6-Fc. As TR6 expresses in many gastrointestinal tumors (Ref.
17 and our unpublished observations), and 
10%
of human intestinal intraepithelial T cells are T cells, we
decided to examine the effect of TR6 on CTL activity of T cells.
For this purpose, Daudi cells, which are known to elicit massive
expansion of V9V2 T cells and are recognized in a TCR-dependent
fashion by these T cells (24, 25, 26), were used as both
stimulators and targets. As shown in Fig. 5A, CTL activity was
enhanced by soluble TR6-Fc but not normal IgG, in agreement with the
proliferation and cytokine studies shown in Figs. 1 and 2. We further
demonstrated that soluble LIGHT significantly inhibited unmanipulated
(without involvement of TR6) CTL activity (Fig. 5B),
suggesting the importance of LIGHT reverse signaling in a CTL
response. Moreover, solid-phase TR6-Fc similarly enhanced CTL
activity, like soluble TR6-Fc, and such enhancement could be
neutralized by soluble LIGHT (Fig. 5C), suggesting that
LIGHT is the likely molecule that TR6 interacts with, and that
cross-linking LIGHT costimulates T cells in CTL development. We
also demonstrated that solid-phase TR6 could augment T cell CTL,
using T cell blasts as target cells (Fig. 5D). The
revelation of such effect of TR6 under physiological conditions is
discussed later.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were initially perplexed by the finding that soluble TR6-Fc enhanced
proliferation and cytokine production in suboptimally
mitogen-stimulated PBMC. Is this because TR6 cross-reacts with other
members of the TNF family and thus blocks their putative negative
effect on T cells via their receptors? So far, no TNFR members,
including Fas and TR2, are known to transduce negative signals into T
cells to inhibit their activation. Therefore, no matter how well
TR6-Fc cross-reacts with other TNF members, no positive signals will be
generated. Thus, we are left with a more plausible mechanism: TR6-Fc
cross-links its ligand on the T cell surface, and the ligand reversely
transduces costimulating signals into T cells. In this model, soluble
TR6, although a TNFR member functions as a ligand, while its ligand
functions as a receptor. How plausible is this reverse signaling
hypothesis? We established a more clear-cut system by putting TR6 on
the solid phase to see whether it could trigger T cell activation. In
this system, the possibility that TR6 functioned as a blocker to
repress the putative inhibitory interactions between any TNF members
and TNFR members was excluded, as TR6 was not in solution, and its
total amount was minute. In the presence of TCR cross-linking (e.g.,
PHA in Fig. 1E, anti-CD3 in Fig. 2B,
anti-TCR in Fig. 4, or alloantigens in Fig. 5, C
and D), solid-phase TR6-Fc augmented T cell proliferation,
lymphokine production, and CTL development, confirming that TR6 can
reversely and directly costimulate T cells. Soluble LIGHT inhibited
TR6-enhanced proliferation (Fig. 1E) and lymphokine
production (Fig. 2B), and repressed CTL development (Fig. 5, C and D). Moreover, LIGHT inhibited the binding
between TR6 and activated Th1 or Th2 cells (Fig. 3). It is acknowledged
that the use of soluble LIGHT as a blocker cannot prove convincingly
that LIGHT mediates the reverse signaling, because one could reasonably
argue that soluble LIGHT has blocked the binding site of TR6 to FasL,
which is also capable of reverse signaling (27, 28, 29). To
address this concern, we used solid-phase TR6 to costimulate
gld/gld mouse T cells, which have mutated nonfunctional
FasL, in the presence of solid-phase anti-CD3. At 0.5 µg/ml of
anti-CD3, wild-type and gld/gld spleen cells similarly
augmented proliferation in the presence of TR6; although at
lower anti-CD3 concentrations, gld/gld spleen cells
responded somewhat less well. We are not sure whether such a difference
is due to the involvement of FasL, but it is obvious that TR6 could
significantly costimulate T cells in the absence of functional FasL.
Moreover, TR2, which binds LIGHT but not FasL, could also costimulate
mouse T cells in the presence of suboptimal anti-CD3. Our most
recent study revealed that the major cell surface ligand that TR6 binds
was LIGHT, because TR6 bound well to wild-type T cells but lost most of
such binding in LIGHT gene knockout T cells (data not shown). Taken
together, these data suggest that a TR6 ligand on the T cell surface
can reversely receive costimulation signals from its receptors, and
that at least a major portion of such costimulation is via LIGHT. With
that said, we cannot rule out the possibility that a small fraction of
the reverse costimulation might also be mediated by FasL, or other so
far uncharacterized TR6 ligand(s) on the T cell surface. As TL1A, the
most recently discovered ligand of TR6, is not expressed on lymphocytes
(19), it is thus unlikely to be involved in such reverse
signaling.
Reverse signaling through ligands is not a far-fetched concept. Several
TNF members on cell surfaces can reversely transduce signals into cells
as with LIGHT. Lanier and colleagues (30) and Gray and
colleagues (31) showed that CD40L transduces costimulation
signals into T cells. Wiley et al. (32) reported that CD30
ligand cross-linking activates neutrophils, and Cerutti et al.
(33) showed that such reverse signaling inhibits Ig class
switch in B cells. Reverse signaling through membrane TNF- confers
resistance of monocytes and macrophages to LPS (34).
Cross-linking of TRANCE enhances IFN- secretion by activated Th1
cells (35). Reverse signaling through FasL can promote
maximal proliferation of CD8 cytotoxic T cells (27, 28, 29).
Cross-linking of TRAIL by its solid-phase death receptor 4 increases
IFN- production and T cell proliferation (36). In the
case of CD40L, its ligation results in general protein tyrosine
phosphorylation, Ca2+ influx, and activation of
Lck, protein kinase C, c-Jun N-terminal kinase, and p38
mitogen-activated protein kinase in EL4 thyoma cells (37, 38). TRAIL cross-linking also induces p38 mitogen-activated
protein kinase activation (36). Therefore, it should not
be surprising that LIGHT can also receive signals from its receptors.
The mechanism of signal transduction via LIGHT is unknown at this time.
LIGHT has a short and featureless cytoplasmic tail (7).
Therefore, the signaling will most likely depend on molecules it
associates with.
We recently reported that human TR6-Fc could bind to mouse LIGHT, and
that human TR6-Fc inhibited CTL in vitro and allograft rejection in
vivo in mice (10). In that paper, the proposed mechanisms
of those observations were that TR6 blocked the costimulation from TR6
to TR2, or reversely from TR2 to LIGHT (10), or both,
although at that time, solid evidence of reverse signaling through
LIGHT was not available. Our current findings have fulfilled one of our
initial predictions that the inhibitory effect of human TR6 in the
mouse system should be attributed to TR6’s interference with the
bidirectional costimulation between TR2 and LIGHT. However, how can we
explain the seemingly opposite effects of soluble human TR6-Fc on human
and mouse T cell responses? The Fc portion used in TR6-Fc and TR11-Fc
was mutated to eliminate FcR binding, and TR6-Fc does not bind to
either human or mouse FcR-bearing cells (data not shown). Therefore,
the opposite effects of TR6-Fc cannot be explained by its effective
anchoring, or the lack of it, on FcR in the human vs mouse systems,
respectively. Rather, this might be a result of different affinity of
human TR6 for human and mouse LIGHT. Because TR6-Fc is a dimer (data
not shown), in theory it can play dual roles when added into solution:
as an inhibitor blocking the bidirectional interaction between TR2 and
LIGHT, or as a costimulator cross-linking LIGHT. The former inhibits T
cell response while the latter enhances it. Its final effect should be
the sum of these two opposite functions, which might have different
affinity requirements. In the human system, we believe that higher
affinity between TR6 and LIGHT tips the balance toward costimulation;
in the mouse system, putative lower affinity between human TR6 and
mouse LIGHT does not result in sufficient LIGHT cross-linking, but
might be enough for TR6 to interfere with the binding between TR2 and
LIGHT. Further studies are needed to examine this hypothesis.
When a stimulatory effect of a molecule is found, we always have to
distinguish between two possibilities: the said molecule indeed has a
positive effect, or inhibits an existing negative regulatory loop. Does
TR6 block an existing negative autocrine loop in which LIGHT acts as a
receptor? This possibility is best argued against by the fact that in
our model, solid-phase TR6-Fc could stimulate T cells. When TR6-Fc was
used to coat the plate, although a concentration of 1–2 µg/100
µl/well was used, only a very small fraction of it actually went onto
the plate, and >99.9% of the protein was washed away after the
coating process. Thus, not >2 ng of TR6-Fc was actually coated on a
well. If we consider how small a fraction of this will leak into
solution, it is unlikely such a minute amount of soluble TR6-Fc could
interfere with an autocrine loop. Can TR6-Fc on the solid phase block
an autocrine loop? We are not aware of any example in an experimental
system that this can be achieved. Because a cell is a three-dimensional
entity, solid-phase TR6-Fc can only interfere with a part of the cell
surface that has contact with the well. Therefore, the solid-phase
TR6-Fc cannot prevent the interaction between a putative soluble
suppressive autocrine and LIGHT on most parts of the cell surface that
are not in contact with TR6-Fc. Consequently, most LIGHT molecules on a
cell surface should still receive negative signals from the putative
suppressive autocrine, if there is one. Therefore, it is very difficult
to explain the positive effect of solid-phase TR6-Fc. In addition, if
solid phase TR6-Fc were to interfere with the negative regulatory loop
by leaking itself into solution, TR6 without Fc (likely monomers)
should be more efficient to do so, and consequently enhance T cell
proliferation better than TR6-Fc. This was obviously not the case,
because we showed that TR6 without Fc on solid phase even failed to
effectively augment T cell proliferation (Fig. 1H). The low
efficiency of TR6 without Fc in this experiment might be due to its
monomer format, which is less potent in cross-linking LIGHT than the
dimeric format of TR6-Fc. Data from literature do not support the
negative loop theory. It has been shown that LIGHT transgenic mice
overexpressing LIGHT on T cells have enhanced immune response
(39, 40 , to be detailed in the next paragraph).
This result does not fit to the model in which LIGHT transduces
negative signals into T cells, because if so, the LIGHT transgenic mice
should have suppressed immune response instead. Lastly, there are
4–5 TNF family members capable of transducing signals into cells,
but none of them transduces a negative one. Based on these arguments,
it is concluded that TR6 exerts its effect by stimulating T cells via
LIGHT, but not by interfering with a putative negative autocrine
loop.
As mentioned above, LIGHT overexpression in the T cell compartment in LIGHT transgenic mice results in profound inflammation and development of autoimmune syndromes (39, 40); T cells overexpressing LIGHT have an activated phenotype (39). Probably, such up-regulated immune response of T cells is due to stimulation of TR2/Herpesvirus entry mediator on dendritic cells by T cell-derived LIGHT, and the dendritic cells in turn augment T cell activity; TR2 on T cells can also receive LIGHT stimulation directly from their fellow T cells (40). However, it is entirely possible that overexpressed LIGHT on the T cells receives stimulation reversely from TR2 expressed on other T cells, and such stimulation augments their responsiveness to TCR ligation.
What is the biological significance of reverse signaling through LIGHT?
We found that the reverse signaling through LIGHT preferentially
promotes Th1 but not Th2 cell cytokine production. In agreement with
this finding, a recent report demonstrated that mucosal T cells
overexpressing LIGHT show enhanced Th1 cytokine production
(39). Because CTL differentiation depends on Th1
cytokines, it is not surprising that CTL activity was augmented after
costimulation through LIGHT by TR6-Fc. It is worth mentioning that
soluble LIGHT inhibited CTL activity (without TR6-Fc stimulation) (Fig. 5C). This finding underscores the importance of LIGHT
costimulation in CTL development. Harrop et al. (41)
noticed that at an intermediate concentration LIGHT promotes MLR, but
it fails to do so at a higher concentration (10 µg/ml). Such a
biphased response probably reflects a shift from costimulation
through TR2 by soluble LIGHT to inhibition of TR2-LIGHT
bidirectional costimulation, depending on the concentration of
LIGHT. This result is consistent with our reverse signaling
theory and findings. We speculate that in vivo, the biological function
of endogenous soluble LIGHT, which comes from cell surfaces after
shedding (9), might be stimulatory or inhibitory,
depending on its local concentration and status of aggregation. Because
dendritic cells also express LIGHT, TR2 on the T cell surface might
activate dendritic cells through LIGHT to modulate their APC function.
If so, this will represent a new mechanism for T cell and dendritic
cell interaction and cooperation. In this study, we used recombinant
TR6-Fc as an artificial binding partner for LIGHT. In vivo, molecules
that can trigger LIGHT signaling are probably cell surface TR2 or
LTR. The endogenous TR6 might act as an inhibitor to the
bidirectional costimulation between TR2 and LIGHT, or function as a
costimulating factor to LIGHT, depending on whether it exists as
monomers, or as trimers like other cell surface TNFR family members.
This aspect is worth further investigation. If the endogenous TR6
functions as an inhibitor, then gastrointestinal tumors secreting TR6
will certainly gain survival advantage by interfering with
as well as T cell CTL activities.
Our study revealed a novel phenomenon of reverse signaling through a cell surface TR6 ligand, most likely LIGHT. Increasing cases of such bidirectional signal transduction between receptors and ligands have been found in biological systems. We can take advantage of such a phenomenon by using a soluble ligand (or receptor) to block signaling in both directions and thus modulate biological responses. However, we must be aware that soluble ligands (or receptors) will need to be monomer without aggregation or cell surface anchoring capabilities to be reliable antagonists, or else they will become agonists for one of the directions.
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Acknowledgments |
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Footnotes |
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2 X.W. and J.Z. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jiangping Wu, Laboratory of Transplantation Immunology, Research Center, Center Hospitalier de l’Universite de Montreal, Notre Dame Hospital, University of Montreal, Pavilion DeSeve, Room Y-5612, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4 M1, Canada. E-mail address: jianping.wu{at}umontreal.ca
4 Abbreviations used in this paper: CD40L, CD40 ligand; FasL, Fas ligand; LTR, lymphotoxin receptor; TRANCE, TNF-related activation-induced cytokine.
Received for publication May 2, 2002. Accepted for publication October 7, 2002.
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