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and IL-12 Production by Monocytes and Dendritic Cells1
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Laboratoire de Recherche sur l’Allergie, Université de Montréal, Centre de Recherche Louis-Charles Simard, Montréal, Québec, Canada; and
Unité d’Immunologie Cellulaire, Institut Gustave-Roussy, Villejuif, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 production by monocytes. Indeed,
anti-LAG-3 mAb inhibits both IL-12 and IFN-
production in
IL-2-stimulated cocultures of T cells and autologous monocytes. Soluble
LAG-3Ig fusion protein markedly enhances IL-12 production by monocytes
stimulated with infra-optimal concentrations of sCD40L, whereas it
directly stimulates monocyte-derived dendritic cells (DC) for the
production of TNF- and IL-12, unravelling an enhanced responsiveness
to MHC class II engagemenent in DC as compared with activated
monocytes. Thus similar to CD40L, LAG-3 may be involved in the
proinflammatory activity of cytokine-activated bystander T cells and
most importantly it may directly activate DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and is up-regulated by IL-2 and IL-12 8, 9 . In vivo, it has
been detected in lymphoid tissues 10 and on T cells infiltrating
renal cell carcinoma 11 . The function of LAG-3 has not been investigated thoroughly. LAG-3 seems to have the capacity to exert both effector and receptor activities. It was reported to transduce a positive signal into LAG-3+ NK cells, as indicated by the defective killing of certain tumor targets by NK cells from LAG-3-/- mice 12 . However, blocking LAG-3-MHC class II interactions with a neutralizing anti-LAG-3 mAb fails to inhibit Ag-driven T cell cytotoxicity and MLR, suggesting that LAG-3 does not provide a positive signal in TCR-driven interactions 10 . Conversely, LAG-3 cross-linking on activated T cells before restimulation via the CD3-TCR complex induces a state of unresponsiveness at both proliferation and cytokine production levels 3 . Finally, during T-T cell interaction, MHC class II engagement by LAG-3 was found to provide a negative signal leading to decreased proliferation and cytokine production 6 .
Here, we first report on the role of LAG-3 in the cytokine-driven
activation of T cells cocultured with autologous monocytes in the
absence of TCR/CD3-mediated stimulation. We show that T cells express
LAG-3 and that LAG-3-MHC class II interaction, together with CD40-CD40
ligand (CD40L) interaction, is involved in both IL-12 and IFN-
production. Blocking LAG-3/MHC contact with anti-LAG-3 mAb not only
suppresses the positive signal given to monocytes via MHC class II but
also inhibits T cell response to IL-12. In addition, we show that
recombinant soluble LAG-3 (LAG-3Ig) dose-dependently induces TNF-
production and costimulates sCD40L-induced IL-12 production by
monocytes. Most interestingly, LAG-3Ig also directly stimulates
dendritic cells (DC) to produce both IL-12 and TNF- without
additional stimulatory or costimulatory signal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sLAG-3Ig molecules produced in Chinese hamster ovary cells as described 6 were kindly provided by M. Subramanyam and M. Tepper (Ares Advanced Technology, Randolph, MA). Preparations contained no detectable endotoxin (<15 pg/ml) as determined by the Limulus amebocyte lysate assay (QCL-100; BioWhittaker, Walkerville, MD). The anti-LAG-3 mAb 17B4 was previously described 2 , and the anti-CD3 UCHT-1 was kindly provided by Dr. P. Beverley (University College and Middlesex School of Medicine, London, U.K.). PHA was from Sigma (St. Louis, MO), and the tuberculin purified protein derivative was from BCG Seizo (Tokyo, Japan). The blocking goat anti-human IL-12 Ab and rIL-2 were kind gifts from Dr. M. Gately and F. K. Kahn, respectively (Hoffmann-La Roche, Nutley, NJ). sCD40L and IL-4 were kindly provided by Dr. C. Maliszewski (Immunex, Seattle, WA), and recombinant granulocyte-macrophage CSF (GM-CSF) was provided by Dr. D. Bron (Bordet Institute, Brussels, Belgium).
Purification of human monocytes and generation of monocyte-derived DC
Monocytes were obtained by aggregation of adult PBMCs in the cold 13 and were depleted of T cells by rosetting on 2-aminoethylisothiouronium bromide (AET) -treated SRBC as previously described 14 . The resulting preparations were >95% CD14+ as determined by FACS (Becton Dickinson, Mountain View, CA) using phycoerythrin- (PE-) conjugated anti-CD14 mAb (Ancell, London, Canada). Autologous T cells were purified by rosetting the monocyte-depleted PBMC with AET-SRBC followed by Lympho-Kwik T treatment (One Lambda, Canoga Park, CA).
Human immature DC were prepared as described 15, 16, 17 . Briefly, monocytes were incubated in 6-well culture plates (5 x 106 cells/3 ml per well) in serum-free RPMI 1640. After 1 h, nonadherent cells were removed and adherent cells were cultured in 3 ml of complete RPMI 1640 medium supplemented with 25 ng/ml GM-CSF and 25 ng/ml IL-4. On day 4, one-half of the culture medium was replaced by fresh medium containing GM-CSF and IL-4, and nonadherent cells were harvested on day 7. Upon microscopic analysis, >98% nonadherent cells presented cellular projections. Analysis by FACS revealed that preparations consisted in a homogenous (>96%) population of CD2-, CD14low/-, CD16-, CD40+, CD54+, CD80+, CD83low/-, CD86+, CD115+, and HLA-DR+ large cells, in agreement with previous reports 15, 16, 17 . Less than 1% of CD3+, CD19+, or CD56+ cells could be detected.
Culture conditions
All cultures were performed in complete HB101 medium (Irvine, Santa Ana, CA) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 IU penicillin, 100 µg/ml streptomycin (BioWhittaker), and 10 µg/ml Polymyxin B (Sigma).
For coculture experiments, T cells (106/ml) were incubated
with autologous monocytes (0.2 x 106/ml) and IL-2 (50
IU/ml). Cell supernatant was collected at day 3 and DNA synthesis at
that time was assessed by the addition of [3H]thymidine
(1 mCi/well; Amersham, Arlington Heights, IL) for 6 h
before harvesting the cells and counting incorporated radioactivity by
liquid scintillation. T cell cultures in the presence of IL-1 and IL-12
were performed exactly as described 19 . The anti-LAG-3 mAb 17B4
and its isotype-matched control (anti-CD5, clone OKT1; American
Type Culture Collection, Manassas, VA) were used in culture at 5
µg/ml. Neither anti-CD5 nor a control mAb of the same isotype not
binding to T cells (anti-Rye, prepared in our laboratory) affected
proliferation or cytokine production. When cultured alone, monocytes
(2 x 106/ml) were preincubated overnight in the
presence of GM-CSF (25 ng/ml) and IFN- (500 IU/ml) as previously
reported 18 before stimulation at 106/ml. DC were also
stimulated at 106/ml.
Cytofluorometric analysis
Binding of LAG-3Ig to monocytes and DC was assessed by indirect immunofluorescence. Briefly, preactivated monocytes or DC were incubated with LAG-3Ig (5 µg/ml) in the presence of normal human IgG (300 µg/ml) for 1 h at 4°C. Cells were then stained with biotinylated Goat anti-human IgG (Tago, Burlingame, CA) for 1 h at 4°C followed by PE-labeled streptavidin (Ancell). Expression of LAG-3 and CD40L was assessed using anti-LAG-3 mAb clone 17B4 2 and anti-CD40L mAb clone 90 13 respectively, or isotype matched control mAb (anti-Rye), at 5 µg/ml in the presence of normal human IgG (300 µg/ml) for 1 h at 4°C. The cells were then stained with biotinylated goat anti-mouse IgG (Tago) for 1 h at 4°C followed by PE-streptavidin. Stained cells were analyzed using a FACScan (Becton Dickinson). To assess the purity of cellular preparations, fluorochrome-coupled mAbs to CD2, CD16, CD19, CD40, CD54, CD56, CD86, and isotype-matched negative controls (all from Ancell) were used; mAb against CD80 and CD83 mAbs were from Immunotech (Coulter, Burlington, Ontario, Canada). The rat anti-CD115 was used in tandem with FITC-coupled goat anti-rat IgG (both from Zymed, San Francisco, CA).
Cytokine measurement
TNF- was measured by two-site sandwich ELISA, and IFN- was
determined by a solid phase RIA as previously described 20 . IL-12 was
detected as previously reported 18 using Ab kindly provided by Dr. M.
Gately (Hoffmann-La Roche). The anti-IL10 assay has been previously
described 21 , and anti-IL-10 rat mAb were obtained from American
Type Culture Collection.
Statistical analysis
Wilcoxon’s test was performed using the Instat Software (GraphPad, San Diego, CA), where * = p < 0.05 ** = p < 0.005.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, LAG-3 was found to be expressed on
TCR-activated T cells. In the experiments summarized in Fig. 1
, primary T cells isolated from blood of
healthy donors were cocultured with autologous monocytes in the
presence of exogenous IL-2 (50 U/ml) and the expression of LAG-3 was
monitored daily by flow cytometry. LAG-3 is absent from resting T
cells, becomes detectable after 24 h of culture, and is
expressed on 60% of the cells at day 7 (Fig. 1, A and
B). LAG-3 is expressed on T cells and not on monocytes as
revealed by two-color analysis (data not shown). Interestingly, the
level of LAG-3 on IL-2-stimulated T cells is not increased by the
addition of PHA (Fig. 1, A and C). Thus LAG-3 may
be expressed at high levels in the absence of TCR signaling. Optimal
induction of LAG-3 is dependent on both exogenous IL-2 and endogenous
IL-12; indeed, IL-2 induces low levels of LAG-3 on purified T cells in
the absence of monocytes, and anti-IL-12 significantly reduces its
expression (Fig. 1D).
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by a mechanism involving CD40-CD40L interaction and
IL-12 production 13 . To examine the role of LAG-3 in this
TCR-independent T cell activation, cultures were supplemented with
neutralizing anti-LAG-3 mAb. As seen in Fig. 2, anti-LAG-3 inhibits T cell
proliferation (42% suppression) and the production of both IFN-
(83% suppression) and IL-12 (56% inhibition). Anti-CD5 mAb, used as
an IgG1 isotype control that also binds to T cells, has no effect.
Anti-LAG-3 mAb does not inhibit IL-12 production by down-regulating
CD40L expression or by inducing the release of IL-10, which remained
undetectable (data not shown). Therefore, it was possible that
anti-LAG-3 suppressed IL-12 production by inhibiting the
interaction between LAG-3 on T cells and MHC class II molecules on
monocytes. Indeed, ligation of MHC class II on murine DC by means of
Abs was shown to stimulate IL-12 production 22 .
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production results only from IL-12 inhibition, we next examined
whether it could be over-ridden by exogenous IL-12. These experiments
(Fig. 3A) reveal that in
addition to suppressing IL-12 production, anti-LAG-3 mAb also
inhibits the response of T cells to IL-12 (51% suppression). This
effect is confirmed by the finding that anti-LAG-3 mAb inhibits
IFN- production (69% suppression) by highly purified
CD4+ T cells stimulated with both IL-12 and IL-1 (Fig. 3B).
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and costimulates IL-12 production
by monocytes
To directly examine whether LAG-3 may regulate or stimulate
cytokine production by monocytes, these cells were first allowed to
up-regulate MHC class II molecules by overnight culture in the presence
of GM-CSF and IFN- 23 . As seen in Fig. 4, the cells express elevated levels of
MHC class II that correlate with a strong capacity to bind LAG-3Ig.
Such activated monocytes were then stimulated with either sCD40L,
LAG-3Ig, or both. As expected, sCD40L induces the production of both
TNF- and IL-12 (Fig. 5, A
and B), whereas LAG-3Ig stimulates only the release of
TNF-. However and most interestingly, LAG-3Ig markedly costimulates
IL-12, as well as TNF-, production in response to suboptimal
concentrations of sCD40L, and this effect is dose-dependent (Fig. 5B). The enhancing effect of LAG-3Ig on sCD40L-induced IL-12
production is observed only when sCD40L is used at an infra-optimal
concentration (Fig. 6). The ability of
LAG-3Ig to markedly enhance IL-12 production in response to suboptimal
but not optimal sCD40L stimulation may explain the suppressive effect
of anti-LAG-3 mAb on IL-12 production in IL-2-stimulated cocultures
of CD4+ T cells and autologous monocytes. Indeed, in these
cultures T cells express very low, albeit functionally significant,
levels of CD40L 13 . These observations may also account for the
finding that the same anti-LAG-3 mAb fails to inhibit IFN-
production when T cells are activated with Ag or mitogen (Table I), stimuli known to induce high levels
of CD40L expression 24 . These effects of LAG-3Ig on monocytes are
specific inasmuch as they are prevented (75%) by preincubation of
LAG-3Ig with anti-LAG-3 mAb (Table II).
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Because DC are an important source of IL-12 and express high
levels of MHC class II, we next examined the ability of LAG-3Ig to
stimulate IL-12 production by monocyte-derived DC. Whereas LAG-3Ig
binds similarly to monocyte-derived DC and to activated monocytes (Fig. 4
), LAG-3Ig directly and dose-dependently induces the production of
IL-12 as well as TNF- by monocyte-derived DC (Fig. 7, A and B) and
costimulates the production of these cytokines, induced by a low dose
of sCD40L. These effects of LAG-3Ig on DC are specific inasmuch as they
are prevented (95%) by the preincubation of LAG-3Ig with
anti-LAG-3 mAb (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN- are also implicated in
Th1-mediated chronic inflammatory diseases 25 . These are presumably
initiated by Ag-specific T cells, and recent evidence 26 further
suggests that they may be amplified and perhaps also perpetuated by
bystander T cells of unrelated Ag specificity that constitute the
majority of the lesional T lymphocyte infiltrate. Our previous in vitro
studies have suggested a mechanism accounting for the activation of
bystander T cells. Resting peripheral blood T cells were shown to be
stimulated for proliferation and IFN- production during coculture
with autologous monocytes in the presence of either IL-2 or IL-15, a
non-T cell-derived cytokine that is produced at inflammatory sites.
Such TCR-independent T cell activation is dependent upon endogenous
IL-12 and TNF- production by monocytes that in turn requires the
engagement of monocyte-associated CD40 with T cell-associated CD40L.
The latter was shown to be expressed at very low but functionally
sufficient levels on freshly isolated primary T cells in the
absence of TCR/CD3 signal 13 . The present results show that LAG-3 is
expressed on cytokine-activated "bystander" T cells and that
it regulates their proinflammatory activity. Addition of
neutralizing anti-LAG-3 mAb suppresses both endogenous production
of IL-12 and T cell response to IL-12. The ability to block IL-12
production by either anti-LAG-3 or anti-CD40L mAbs 13
suggests that the LAG-3/MHC class II and CD40/CD40L signaling pathways
act in synergy to induce IL-12 production in this culture system. This
view is supported by the finding that LAG-3Ig markedly costimulates
IL-12 production in response to suboptimal concentrations of sCD40L
trimer and allows the production of IL-12 in response to suboptimal
sCD40L stimulation. In contrast to IL-12, the production of TNF- is
directly triggered by LAG-3Ig without CD40L stimulation, underlining
the differential regulation of these cytokines. In addition to their
well-defined functions as peptide-presenting structures and response
restriction elements, MHC class II molecules can also transduce signals
in the APCs on which they are expressed 27, 28 . Cross-linking of MHC
class II molecules has been reported to up-regulate TNF- expression
29, 30, 31, 32 as well as IL-1, IL-6, and nitric oxide production by
monocytic cells 30, 33, 34 and to induce IL-12 production by murine
DC 22 . Most interestingly, the enhancing effect of LAG-3Ig on IL-12
production is observed only with suboptimal, and not with optimal,
CD40L stimulation. This is consistent with the observations (Ref. 10
and Table I) that anti-LAG-3 mAb does not suppress IFN-
production by Ag- or mitogen-stimulated T cells, which express much
more CD40L than cytokine-activated cells 24 . Addition of exogenous
IL-12 only partly overcomes the suppressive effect of anti-LAG-3
mAb on IFN- production in the T cell/monocyte coculture system,
indicating that ligation of T cell-associated LAG-3 inhibits the
response to IL-12. This inhibition might result from two mechanisms.
First, the blockade of LAG-3-MHC class II interaction may inhibit
response to IL-12; this view implies that engagement of
LAG-3 by its ligand expressed on monocytes delivers a positive signal
to T cells, allowing them to respond to IL-12. Alternatively, LAG-3
ligation may provide direct inhibitory signal to T cells. The second
mechanism is supported by the finding that anti-LAG-3 suppresses
the response of highly purified T cells to IL-12 plus IL-1. Indeed,
this cytokine mixture does not induce the expression of class II
molecules on T cells. This view is further supported by the previous
findings that ligation of LAG-3 before TCR/CD3 stimulation markedly
inhibits T cell proliferation and cytokine production 3 . Taken collectively, the present data are consistent with the concept that LAG-3 is a two-way signaling molecule, providing a negative signal to the cells on which it is expressed and a stimulatory signal to those expressing its ligand, MHC class II. Our results further show that the stimulatory activity of LAG-3 on monocytes differs from that on monocyte-derived DC. Indeed, unlike in monocytes, LAG-3Ig directly stimulates IL-12 production by DC without the need of CD40L signal. This differential effect of LAG-3 cannot be explained by the differential expression of MHC class II molecules on these two types of APCs. Indeed, the preactivated monocytes employed in our study expressed similar levels of HLA-DR as the DC. The ability of LAG-3Ig to directly stimulate IL-12 production by DC is consistent with its protective effect on experimental tumor growth.4 In that study, coinjection of LAG-3Ig together with wild-type tumor cells (or injection of tumor cells transfected with LAG-3) inhibits tumor expansion and confers T cell-dependent protection against rechallenge with wild-type tumor.
In conclusion, the present results indicate that LAG-3, which is
typically expressed on TH1-like cells 8 , stimulates APC for increased
TNF- and IL-12 production. They further suggest that some
anti-LAG3 mAbs have the profile of anti-inflammatory agent
capable of blocking the activation of bystander proinflammatory T
cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christian E. Demeure, Laboratoire de Recherche sur l’Allergie, Université de Montréal, CHUM Pavillon Notre-Dame, porte M4211-K, 1560 rue Sherbrooke Est, Montréal, Québec, H2L 4 M1, Canada. E-mail address:
3 Abbreviations used in this paper: LAG-3, lymphocyte activation gene-3; LAG-3Ig, LAG-3 immunoadhesin; DC, dendritic cells; GM-CSF, granulocyte-macrophage CSF; PE, phycoerythrin; CD40L, CD40 ligand; s, soluble.
4 P. Prigent, C. Demeure, S. El Mir, S. Hannier, M. Tournier, M. Dreano, G. Delespesse, and F. Triebel. 1998. LAG-3 induces tumor regression and anti-tumor immune responses in vivo. Submitted for publication.
Received for publication July 21, 1998. Accepted for publication December 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion and triggering of tumor necrosis factor release. J. Exp. Med. 180:1005.. J. Exp. Med. 179:1109. by neonatal human CD4 T cells. J. Immunol. 151:1938.[Abstract]
increases HLA-DR synthesis and expression. J. Immunol. 130:1492.[Abstract]
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