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by Human V
9V
2 T Cells Via Engagement of FcRIIIA, the Low Affinity Type 3 Receptor for the Fc Portion of IgG, Expressed upon TCR Activation by Nonpeptidic Antigen1
Institut National de la Santé et de la Recherche Médicale, Unité 431, Microbiologie et Pathologie Cellulaire Infectieuse, Université Montpellier II, Montpellier, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR represent a minor T
cell subpopulation found in blood. The majority of these cells express
V9V2 determinants and respond to nonpeptidic phosphoantigens.
Several studies have shown that, in vivo, the percentage of V9V2
T cells dramatically increases during pathological infection, leading
to the hypothesis that they play an important role in the defense
against pathogens. However, the specific mechanisms involved in this
response remain poorly understood. It has been established that
V9V2 T cells display potent cytotoxic activity against
virus-infected and tumor cells, thereby resembling NK cells. In this
study, we show that, upon stimulation by nonpeptidic Ags, V9V2 T
cells express FcRIIIA (CD16), a receptor that is constitutively
expressed on NK cells. CD16 appears to be an activation Ag for
V9V2 T cells. Indeed, ligation of CD16 on V9V2 T cells
leads to TNF-
production. This TNF- production, which is
dependent (like that induced via the TCR-CD3 complex) on the activation
of the p38 and extracellular signal-regulated kinase-2
mitogen-activated protein kinases, can be modulated by CD94 NK
receptors. Therefore, it appears that V9V2 T cells can be
physiologically activated by two sequential steps via two different
cell surface Ags: the TCR-CD3 complex and the FcRIIIA receptor,
which are specific cell surface Ags for T lymphocytes and NK cells,
respectively. This strongly suggests that, in the general scheme of the
immune response, V9V2 T cells represent an important
subpopulation of cells that play a key role in the defense against
invading pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR represent a minor T cell
subpopulation in blood and peripherallymphoid organs (reviewed in
Refs. 1, 2). The majority of these T cells express
the V9V2 TCR and are
CD3+CD4-CD8-
(3). In vivo, their percentage dramatically increases
during infection by intracellular pathogens of viral, bacterial, and
parasitic origin (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). A particular feature of these
V9V2 lymphocytes is that they respond to nonpeptidic
phosphoantigens that are not processed and presented in a
MHC-restricted manner (15, 16, 17, 18, 19). Recently, they have been
shown to also respond to alkylamines with structures as simple as
ethylamine (20). Following stimulation by nonpeptidic
ligands, mature V9V2 T cells proliferate, release large amounts
of cytokines, and acquire cytotoxic activity against tumor cells
(19) or virus-infected cells (21). An
important mechanism controlling lymphoid cell activation involves the
so-called MHC class I-specific inhibitory receptors, which were
initially characterized on NK cells but were recently found on 
and T lymphocytes as well. Two distinct classes of inhibitory
receptors have been described. One includes members of the Ig
superfamilly (p58, p70, and p140) that interact with particular HLA-A,
HLA-B, or HLA-C alleles (22). The other, formed by a
covalent assembly of C-type lectins (CD94 and NKG2A), recognizes HLA-E,
a nonclassical class I molecule (23). A large proportion
of circulating V9V2 T cells express inhibitory receptors that
belong mostly to the C-type lectin family (CD94-NKG2)
(24). The interaction between MHC class I molecules and
CD94-NKG2 receptors down-modulates antigenic activation of T
cells by interfering with the TCR signaling cascade (25),
thereby inhibiting T cell proliferation and cytokine production
in response to microbial phosphorylated ligands (25, 26, 27).
The interaction between MHC class I molecules and CD94-NKG2A receptors
also controls the cytolytic activity of peripheral V9V2 T cells
and V9V2 T cell clones (27, 29) against tumor and
virus-infected cells. Therefore, these receptors, through their
interaction with MHC class I molecules, allow T cells (as is the
case for NK cells) to carry out immunosurveillance for "missing
self" (27). In this regard, V9V2 T cells resemble
NK cells.
NK cells express FcRIIIA (CD16) (30, 31), the low
affinity type 3 receptor for the Fc portion of IgG, which has been
defined as a transmembrane protein with a cytoplasmic domain that
associates with hetero- or homodimers of the 
(CD3)- and
(Fc
RI)-chains (32, 33). Several studies have
demonstrated that ligation of CD16 stimulates cytotoxicity and cytokine
secretion (34, 35, 36). Moreover, it was shown that
cross-linking of CD16 on NK cells resulted in increased intracellular
calcium levels and in a cascade of biochemical events similar to those
activated in lymphocytes via the TCR (37, 38, 39, 40, 41, 42, 43). CD16
is known to be involved in Ab-dependent cellular cytotoxicity;
however, an additional role for CD16 on human NK cells as a lysis
receptor was recently suggested. Indeed, CD16 was found to mediate the
direct killing of some virus-infected and tumor cells independent of Ab
ligation (44).
Previous studies have reported that T cells express CD16 Ag
(45, 46), but neither of these studies specified the
subset or the state of activation of the CD16-positive T cells
used. In the present study, we have analyzed the expression of CD16 on
V9V2 T cells according to their activation state. Activation was
induced by different mitogens, including the mycobacterial Ag
isopentenylpyrophosphate
(IPP),3 a nonpeptidic
compound that specifically stimulates V9V2 T cells. We show that
expression of CD16 is induced after TCR stimulation and lasts for at
least 4–6 wk. As with NK cells, cross-linking of CD16 by immobilized
anti-CD16 mAb or IgG-coated cells triggers V9V2 T cells to
release high levels of TNF-. This TNF- production via CD16 is
dependent, like that induced via TCR-CD3, on the activation of the p38
and extracellular signal-regulated kinase (ERK)-2/mitogen-activated
protein kinase (MAPK) pathways. However, when we compare the overall
phosphotyrosine signaling induced either through ligation of TCR-CD3 or
CD16, it appears that the two corresponding electrophoresis profiles
are different, demonstrating that the phosphorylation pathways are
differentially activated according to the Ag involved. Moreover, we
provide evidence that TNF- production induced through CD16 ligation
can be modulated through NK receptors. Taken together, these results
suggest that, once stimulated directly through the TCR-CD3 complex by
nonpeptidic molecules released by a pathogen, V9V2 T cells
acquire, through long-lasting CD16 expression, the ability to be
reactivated by Abs produced during the acquired immune response,
leading to further production and release of large amounts of
TNF-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant IL-2 was purchased from Chiron (Emeryville,
CA), IPP from Sigma (St. Louis, MO), and SB 203580 from Calbiochem
(Nottingham, U.K.). PD 98059, anti-phospho p38 MAPK Ab,
anti-p38 MAPK Ab, anti-phospho p42/44 MAPK Ab, anti-phospho
Elk-1 Ab, anti-Elk-1 Ab, anti-phospho activating transcription
factor (ATF)-2 Ab, and anti-ATF-2 Ab were all purchased from
New England Biolabs (Beverly, MA). Anti-ERK-2 Ab was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine Ab
(4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).
HRP-conjugated anti-mouse Ab and anti-rabbit Ab were from
Amersham Pharmacia Biotech (Paris, France). UCHT1 (anti-CD3 mAb,
IgG1), two anti-CD4 mAbs (BL4, IgG2a; and 13B8.2, IgG1),
anti-TCR V2 (IgG1), anti-TCR V9 (IgG1), anti-TCR pan
(IgG2b), anti-CD16 (IgG1), anti-CD56 (IgG1),
anti-CD69 (IgG2a), anti-CD94 (IgG2a) mAbs, and isotypically
matched control mouse IgG1 and IgG2a (conjugated or not) were all
purchased from Immunotech (Marseille, France).
Isolation and cell culture
PBMC were isolated from healthy donors. Human peripheral
blood-derived T cells were generated using the following
procedure: T cells were purified from PBMC by positive
immunoselection using anti-TCR V9 mAb in conjunction with
magnetic beads coated with anti-mouse IgG. After spontaneous
detachment, T cells were specifically activated in the presence
of syngeneic monocytes, IPP (50 µM), and rIL-2 (20 ng/ml). Human
peripheral blood-derived T lymphoblasts were generated as
described above and maintained in RPMI 1640 supplemented with 5% FCS,
5% human AB serum, 2 mM glutamine, and rIL-2 (20 ng/ml) at 37°C in
5% CO2-humidified atmosphere for 4 or 5 wk. In
some experiments, T cells were obtained by negative selection
from PBMC from healthy donors as follows: After separation on a Ficoll
gradient, monocytes were separated by adherence on plastic, followed by
the removal of B cells, T cells, and NK cells using the specific
Abs anti-CD19, anti-pan TCR, and anti-CD56 mAbs,
respectively, in conjunction with magnetic beads coated with
anti-mouse IgG. Immediately after purification, T cells were
either fixed with 1% paraformaldhehyde solution or maintained in
culture for 1 wk in the presence of rIL-2 alone (20 ng/ml) or rIL-2 (20
ng/ml) and IPP (50 µM) and were then analyzed by flow cytometry.
Flow cytometry
To block nonspecific binding, cells (0.5 x
106) were incubated with 10% human AB serum for
30 min. T cells were then stained with 1 µg PE -labeled
anti-CD3 mAb and FITC-labeled TCR V2 mAb, 1 µg PE-labeled
anti-TCR V9 mAb and FITC-labeled TCR V2 mAb, 1 µg
PE-labeled anti-CD16 mAb, or 1 µg PE-labeled anti-CD25 mAb.
After CD16+ cells were purified from PBMC by
positive immunoselection, they were stained with FITC-labeled TCR V2
mAb and PE-anti-CD56 mAb. During staining, cells were incubated
with Abs in PBS supplemented with 2% FCS and 0.02%
NaN3, on ice, in a total volume of 50 µl. After
30 min, cells were washed once, fixed in 1% paraformaldehyde solution,
and analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) with
CellQuest software.
Preparation of supernatants for measurement of TNF- production
T cells (1 x 106) were
cultured in 24-well tissue culture plates in RPMI 1640 supplemented
with 5% FCS and 5% human AB serum in a total volume of 0.5 ml/well
and incubated in presence of one of the following stimulatory agents:
anti-CD3 mAb (10 µg/ml), coated anti-CD16 mAb, anti-CD4
(BL4 and 13B8.2 clones; 10 µg/ml), goat anti-mouse Ig (GamIg; 10
µg/ml), soluble anti-CD16 (10 µg/ml), isotypically matched
control mouse IgG1 and IgG2a (10 µg/ml), or
CD4+ T cells coated or not with anti-CD4 mAb
(2 x 106 cells). The coating was performed
using an Ab concentration of 10 µg/ml in 0.1 M carbonate buffer (pH
9.5) for 2 h at 37°C. Syngeneic CD4+ cells
were purified by positive immunoselection using magnetic beads coated
with anti-CD4 mAb (Dynal Biotech, Oslo, Norway) according to the
manufacturer’s instructions. After purification, cells were fixed in
1% paraformaldehyde-PBS solution. CD4+ cells
were washed twice with PBS and incubated with or without anti-CD4
mAb (BL4 or 13B8.2; 10 µg/ml) for 30 min, followed by a second wash
before adding them to the T cells. When mentioned, T
cells were pretreated with specific pharmacological inhibitors of p38
and ERK-2/MAPK pathways (SB 203580 (20 µM) or PD 98059 (20 µM),
respectively) for 30 min at 37°C. Supernatants were harvested and
assayed for TNF- using the OptEIA human TNF- ELISA kit (BD
PharMingen, San Diego, CA) according to the manufacturer’s
instructions.
Inhibition of TNF- production by CD94
T cells (1 x 106) were
incubated at 4°C for 30 min with anti-CD94 mAb (10 µg/ml),
anti-CD69 mAb (10 µg/ml), or isotypically matched control mouse
IgG2a, washed, and then cultured in 24-well tissue culture plates in
RPMI 1640 supplemented with 5% FCS and 5% human AB serum in a total
volume of 0.5 ml/well in the presence of either coated anti-CD16
mAb or GamIg (10 µg/ml). Supernatants were harvested and assayed for
TNF- using the OptEIA human TNF- ELISA kit according to the
manufacturer’s instructions.
Cell extract preparation and Western blot analysis
T cells (20 x 106) were
stimulated by UCHT1 (10 µg/ml) or coated-CD16 (10 µg/ml) at 37°C
for the indicated times. When mentioned, T cells were pretreated
with p38 and ERK-2 inhibitors (SB 203580 (20 µM) or PD 98059 (20
µM), respectively) for 30 min at 37°C. Following stimulation, cells
were lysed in 1 ml lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM
NaCl, 10 mM NaF, 10 mM iodoacetamide, 1% Nonidet P-40, 1 mM PMSF, 1 mM
Na2VO3, and 1 µg/ml of
each protease inhibitor (leupeptin, aprotinin, and chymostatin).
Proteins were concentrated byprecipitation with 1.5 vol of acetone.
Proteins from 5 x 106 cells were separated
by 10% SDS-PAGE, transferred to polyvinylidene difluoride
membranes (Millipore, Bedford, MA), and detected with the relevant Abs:
anti-phospho p38 MAPK Ab (1:1000), anti-p38 MAPK Ab (1:1000),
anti-phospho p42/44 MAPK Ab (1:1000), anti-ERK-2 Ab
(1:5000),anti-phospho Elk-1 Ab (1:1000), anti-Elk-1 Ab (1:1000),
anti-phospho ATF-2 Ab (1:1000), anti-ATF-2 Ab (1:500), or
anti-phosphotyrosine mAb (1:1000). Immunoreactive bands were
visualized using the chemiluminescence Western blotting system
(Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9V2 T
cells
V9V2 T cells were purified from PBMC from healthy donors by
positive immunoselection and cultured in the presence of IPP, syngeneic
monocytes, and rIL-2. After 1 wk of culture, flow cytometry analysis of
cells double stained with anti-CD3/anti-V2 demonstrated that
live T lymphocytes present in the culture medium were >99%
V2-expressing cells (Fig. 1
A). Moreover, a second double
staining using anti-V9/anti-V2 confirmed that these cells
were V9V2 T cells (Fig. 1B). These analyses were
performed for each preparation of human peripheral blood-derived
T cells used in this study.
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9V2 T cells were then analyzed for expression of
FcRIIIA (CD16) as a function of time after stimulation. As shown in
Fig. 2A, CD16 is only
moderately expressed on activated cells after 8 days (low fluorescence)
in the presence of IL-2, but its expression is up-regulated over time
and reaches a plateau after 
3 wk of stimulation (strong
fluorescence). Expression of CD16 is subsequently slowly down-regulated
to an undetectable level 4–6 wk after stimulation. Interestingly, the
increased expression of CD16 does not follow that of CD25, a well-known
activation marker for T cells; indeed, CD25 is expressed early (<8
days) and then diminishes very rapidly, while CD16 expression increases
slowly and reaches its maximum when CD25 has almost disappeared (Fig. 2A). This pattern of CD25 and CD16 expression was
consistently observed in the analyses of human peripheral blood-derived
T cells from at least 10 healthy donors and is schematically
represented in the curves below. CD16 expression was determined shortly
after stimulation (5–8 days); however, we were also interested to know
whether this Ag was expressed on circulating resting V9V2 T
cells, because it has been reported that circulating T cells
might express CD16 Ag (45, 46). Therefore, we analyzed
CD16 expression on freshly isolated unstimulated V9V2 T cells. To
avoid any activation triggered by the anti-9 mAb that we used to
positively select for V9V2 T cells, the total T cell
population was purified by negative selection. This was achieved by the
removal of macrophages, T cells, B cells, and NK cells from PBMC
as described in Materials and Methods. To avoid potential
internalization of CD16, we performed the purification at 4°C and
then either directly fixed the cells in 1% paraformaldehyde solution
or maintained the cells in culture for 1 wk in the presence of rIL-2
alone or rIL-2 and IPP. Flow cytometry analysis of the purified
populations using a double staining with anti-2 and
anti-CD16 mAbs showed that the V2-expressing cells were not
stained by anti-CD16 mAb, indicating that CD16 is not coexpressed
on unstimulated cells in the presence or absence of IL-2 (Fig. 2, B and C, left panels). However,
following stimulation by IPP and 1 wk of culture, 40% of
V2-expressing cells were stained by anti-CD16 (Fig. 2C, right panel). Moreover, CD16-positive cells
purified by positive selection using an anti-CD16 mAb and
anti-IgG coated magnetic beads were not stained by anti-V2
mAb, whereas 80% were stained by anti-CD56 mAb, which
recognizes CD56, a specific marker for NK cells (Fig. 2B,
right panel). These results demonstrate that CD16 can be
considered as a marker of stimulation of V9V2 T cells.
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RIIIA-induced stimulation of V9V2 T lymphocytes
Therefore, we questioned whether CD16 Ag, which is expressed after
TCR-induced stimulation, can act as a stimulatory Ag. In NK cells, CD16
is an efficient stimulatory Ag, and its ligation and cross-linking
triggers cytotoxicity, expression of activation markers such as CD25,
as well as production of cytokines such as IFN- and TNF-
(35, 36). Because stimulation of V9V2 T cells via
the TCR leads to cytokine production, in particular TNF- release
(47), we studied whether V9V2 T cells can produce
this cytokine upon CD16 ligation. V9V2 T cells that strongly
expressed CD16 and were stimulated for 6 h with either
anti-CD10 mAb or anti-CD3 mAb were used. Fig. 3A demonstrates that, when
CD16 is cross-linked by anti-CD16 mAb coated to plastic wells,
V9V2 T cells are triggered to produce TNF-. The amounts of
TNF- produced upon CD16 stimulation are at least equivalent to those
produced upon stimulation by anti-CD3 mAb (or IPP; data not shown)
through the TCR, whereas isotypically matched control IgG1 Ab did not
induce TNF- production. In addition to the observed up-regulation of
CD16 on activated V9V2 T cells (Fig. 2
A), we also
noticed that the level of TNF- production was closely related to the
density of CD16 Ag present on the cell membrane. Indeed, as shown in
Fig. 3B, there is a very low cytokine release by T
cells weakly expressing CD16 (CD16+) in contrast
to those strongly expressing CD16 (CD16+++). In
addition, a decrease of cytokine production to an undetectable level
was observed after 4–6 wk (data not shown).
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9V2 T cells by IgG-coated cells
Because FcRIIIA is a low affinity receptor for IgG Fc fragment,
we tested whether V9V2 T cells could be activated through this
receptor by a physiological model (i.e., cells previously coated with
IgG). Therefore, V9V2 T cells were cultured in the presence of
syngeneic formaldehyde-fixed purified CD4+
T cells that were previously coated (or not) with anti-CD4 mAb. As
shown in Fig. 4, anti-CD4-coated
cells induced TNF- production by T cells. No cytokine
production was observed in the presence of uncoated
CD4+ T cells or anti-CD4 mAb alone. It should
also be noted that TNF- production was higher when
CD4+ cells were coated with anti-CD4 of the
IgG2a isotype (BL4 clone) rather than mAb of the IgG1 isotype (13B8.2
clone). This result could suggest that FcRIIIA on V9V2 T cells
displays a better affinity for IgG2a than IgG1, as is the case for CD16
Ag expressed by NK cells (36).
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was indeed
mediated via CD16, blocking experiments with soluble anti-CD16 mAb
were performed (anti-CD16 mAb is not stimulatory unless immobilized
on a solid support or cross-linked by GamIg). As shown in Fig. 4, addition of soluble anti-CD16 mAb totally abrogated TNF-
production, while addition of an isotypically matched control Ab (mouse
IgG1) did not prevent TNF- production.
TNF- production by CD16-stimulated V9V2 T cells is
dependent on the p38 MAPK activation pathway
Intracellular signal-regulating TNF- production has
been extensively studied in monocytes upon activation by LPS
(48, 49, 50, 51, 52, 53). In these cells, activation of p38 MAPK appears
to be necessary for production of the cytokine, as demonstrated by the
use of SB 203580, a, specific p38 MAPK inhibitor (51). In
T cells of the lineage, TNF- production has been demonstrated
to be dependent on engagement of CD28 (54), but the
involvement of p38 MAPK, whose activation is generally related to this
coreceptor, remains controversial (54, 55, 56). Recently, we
provided evidence that, in V9V2 T cells, TNF- production
triggered via TCR does not involve CD28 as a costimulatory molecule but
requires the activation of the p38 MAPK pathway (47).
Because, in V9V2 T lymphocytes, TNF- release can be triggered
via TCR as well as via CD16, we questioned whether the cytokine
production via CD16 is also regulated by the p38 MAPK activation
pathway. As can be seen in Fig. 5A, SB 203580, a
pharmacological p38 kinase inhibitor, blocked TNF- production by
V9V2 T cells activated via CD3 and CD16, demonstrating that
production of the cytokine is dependent on activation of this kinase.
In subsequent experiments, we determined whether the p38 MAPK pathway
was triggered after stimulation of V9V2 T cells via CD16. In Fig. 5B, we show that phosphorylation of p38 MAPK occurs not only
upon TCR-CD3 complex recruitment, but also after CD16 recruitment. To
correlate the phosphorylation of p38 MAPK with its activity, we studied
the phosphorylation of one of its substrates, ATF-2, after pretreatment
(or not) with SB 203580 compound, the specific inhibitor of p38 MAPK.
As illustrated in Fig. 5C, a phosphorylation of ATF-2 was
observed following both CD3 and CD16 activation, and this
phosphorylation was abrogated by pretreatment with SB 203580. These
results indicate that phosphorylation of ATF-2, observed after CD3 and
CD16 cross-linking, is dependent on p38 kinase activation.
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is dependent on the MAPK kinase
(MEK)/ERK activation pathway
In a previous report, we have also shown that ERK-2 activation is
a necessary step for TNF- production in TCR-induced activated
V9V2 T cells (47). Therefore, we analyzed activation
of this kinase upon engagement of CD16. As shown in Fig. 6A, ERK-2 appears to be
phosphorylated following both TCR and CD16 stimulation. To correlate
the phosphorylation of ERK-2 with its activity, we studied in parallel
the phosphorylation of one of its substrates, Elk-1. As illustrated in
Fig. 6B, Elk-1 was phosphorylated following both CD16 and
CD3 activation, and this phosphorylation was abrogated when the ERK-2
pathway was inhibited by pretreatment with PD 98059, a pharmalogical
inhibitor of MEK-1, the upstream kinase that phosphorylates ERK-2.
Moreover, we showed that activation of this kinase is necessary for
cytokine release through CD16, as is the case in TCR-CD3 stimulation.
Indeed, as shown in Fig. 6C, the addition of PD 98059
completely blocked production of TNF-. Taken together, these data
confirm a dependency on the MEK/ERK pathway for TNF- production by
T cells activated via CD16.
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Thus, TNF- is produced upon stimulation either through TCR-CD3
or CD16 and, in both cases, ERK-2 and p38 MAPK pathways are activated.
Moreover, like the TCR-CD3 complex, CD16 is associated with
immunoreceptor tyrosine-based activation motif-containing chains (
and FcRI), which, through their phosphorylation, can trigger
transducing activating pathways to the nucleus (30, 31, 32, 33).
Therefore, we questioned whether the overall signaling is similar in
the two Ag-induced pathways. To investigate this, we studied the
tyrosine phosphorylation occurring during the two types of activation
at different time points. As can be seen in Fig. 7, the two tyrosine phosphorylation
electrophoresis profiles are significantly different. In particular, a
rapid increase in the tyrosine phosphorylation of several cellular
proteins is observed only 5 min after CD3 activation, whereas 30 min of
CD16 activation is necessary to observe a subsequent increase in
tyrosine phosphorylation of cellular proteins. These results indicate
that, in addition to differences in the protein profiles observed via
tyrosine phosphorylation, the kinetics of the overall signaling
pathways activated by these two different Ags appears to differ.
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9V2 T cells is blocked by the
CD94 recruitment
It is well established that engagement of the inhibitory complex
CD94-NKG2A by natural MHC class I ligand-bearing cells or by specific
Abs inhibits important pathways leading to CD16-triggered cytotoxicity
in NK cells (57, 58). Moreover, CD94-NKG2A is expressed by
the majority of V9V2 T cells (26, 59 and data not
shown), and it has been demonstrated that engagement of this complex
induces a down modulation of TCR-CD3 complex-induced activation.
Therefore, we questioned whether TNF- production induced in
V9V2 T cells through CD16 could be modulated via CD94. To address
this question, we used anti-CD94 mAb to interact with CD94 and
mimic the interaction with MHC class I molecules as described elsewhere
(28). We first incubated V9V2 T cells with
anti-CD94 mAb or an isotypically matched control Ab (IgG2a) for 30
min at 4°C and then maintained the cells in culture for 6 h in
the presence of coated anti-CD16 with or without GamIg. Fig. 8 shows that TNF- production induced
via CD16 by anti-CD16 mAb is largely inhibited upon treatment with
anti-CD94 and GamIg, suggesting that CD16-induced activation of
V9V2 T cells can be regulated by CD94-NKG2 engagement. It should
be noted that inhibition of TNF- production only occurs when both
CD94 and CD16 are cross-linked by GamIg through their respective mAbs
(i.e., anti-CD94 mAb alone does not retain the ability to block
production of the cytokine). Moreover, cross-linking of an irrelevant
surface Ag such as CD69 (using an anti-CD69 mAb with the same
isotype as anti-CD94 mAb (IgG2a)) together with CD16 Ag did not
modify the production of TNF- induced by coated anti-CD16 Ab.
Therefore, the inhibition of TNF- production induced via CD16
appears to be specific to cross-linking of this Ag with CD94.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9V2
T cells via the TCR-CD3 complex by anti-CD3 mAb or the
mycobacterial phosphoantigen IPP leads to TNF- production
(47). In the present work, we show that, once activated
through the TCR, V9V2 T cells express FcRIIIA (CD16),
the low affinity type 3 receptor for IgG Fc fragment. Interestingly,
CD16, which represents a TCR-mediated activation marker, is not early
and rapidly expressed after stimulation like other activation markers,
such as CD25 or CD69 (data not shown), but is expressed at a later
stage, and its expression increases over time.
This receptor has been well described as a cell surface Ag
constitutively expressed on NK cells and is known to mediate
Ab-dependent cellular cytotoxicity as well as cytokine
production,including TNF-, in these cells (30, 31, 36). In this study, we have demonstrated that, when
cross-linked, this cell surface Ag is able to activate V9V2 T
cells, leading to TNF- production. It is noteworthy that the primary
activation triggered by IPP occurs via the TCR-CD3 complex, and in this
respect, the cells behave as normal T cells. However, once activated,
the V9V2 T cells acquire through CD16 expression an NK cell
character and, as is the case for NK cells, acquire the ability to be
reactivated through engagement of this Ag. Therefore, it appears that
V9V2 T cells can be physiologically activated in two sequential
steps via two different cell surface Ags: the TCR-CD3 complex and the
FcRIIIA receptor, which are specific cell surface Ags for T
lymphocytes and NK cells, respectively. Using
CD4+ T cells previously coated with mouse
anti-CD4 mAb, we demonstrated that V9V2 T lymphocytes can be
activated by these anti-CD4-coated cells through IgG-Fc
fragment-CD16 interaction. Taken together, these results lead to the
hypothesis that, during the process of a pathogenic infection,
V9V2 T cells can be activated early by nonpeptidic Ags released
from the pathogen, which leads, on one hand, to direct cytokine
production including TNF- and IFN-, and on the other hand, to
cell surface expression of CD16. Once this latter Ag is expressed,
V9V2 T cells can behave as NK cells and can be reactivated later,
via this Ag, by Ab-coated infected cells, leading to further production
of cytokines. This scenario may occur in the case of a viral infection,
in which viral components are expressed on the surface of the infected
cell (60), and this may also be the situation in bacterial
disease. Indeed, it has been demonstrated that during infection with
Brucella, LPS from the bacteria is expressed on the cell
surface of infected macrophages (61). In parallel, several
studies have shown that, during Brucella infection, there is
a strong Ab response directed against Brucella LPS
(62). Moreover, we recently demonstrated that
Brucella suis bacteria produce a nonpeptidic fraction that
specifically stimulates V9V2 T cells, leading to high production
of TNF- and IFN- (63) and also to CD16 expression
(data not shown). Interestingly, when V9V2 T cells express CD16
and acquire the ability to produce cytokines through this Ag, they do
not lose the capacity to be stimulated through the TCR-CD3 complex.
Therefore, at this stage, V9V2 T cells can be activated through
both TCR-CD3 and CD16 cell surface Ags to produce TNF-. One can
question whether production of TNF- by these cells through the two
different Ags involves two different signaling routes. We have
demonstrated that, in both cases, TNF- production requires
activation of the ERK-2 and p38 MAPK pathways. In addition, it is known
that the TCR as well as CD16 are associated with immunoreceptor
tyrosine-based activation motif-expressing chains (i.e., -chain for
both Ags and also FcRI for CD16) (32, 64). However, the
overall signaling, particularly tyrosine phosphorylation, appears to
differ upon stimulation via the two different Ags. This suggests that,
if TNF- is a common distal biological event in the two stimulation
processes, other differentially regulated events could be triggered by
the two cell surface Ags.
As previously shown (26, 27, 59), we also confirmed that
V9V2 T cells constitutively express CD94, the C type lectin
family NK receptor (data not shown). Engagement of this receptor by
anti-CD94 mAb was demonstrated to inhibit V9V2 T cell
proliferation and cytokine production (TNF- and IFN-) in response
to TCR-mediated mycobacterial phosphoantigen activation (26, 27). This implied that direct engagement of CD94 was sufficient
to trigger signals that directly inhibit TCR-induced signaling.
Therefore, we questioned whether CD94 is also able to modulate
activation of V9V2 T cells induced through CD16. We demonstrated
that anti-CD94 mAb is indeed able to inhibit TNF- production
induced upon CD16 binding; however, in this case, CD94 has to be
engaged by the binding of anti-CD94 mAb and must also be
cross-linked with CD16. Indeed, when anti-CD94 is added alone,
TNF- produced upon anti-CD16 binding is not modified. This is in
contrast to what was observed in TNF- production induced via TCR,
where engagement of CD94 is sufficient to trigger inhibition of TNF-
release. This difference is possibly related to the fact that CD94 is
not far from the TCR-CD3 complex and can therefore modulate activation
signals triggered at the neighboring TCR/CD3 complex through associated
enzymes (likely Src homology 2 domain-containing tyrosine phosphatase,
SHP-1). In the case of CD16 activation, CD94 could need to be
brought close to CD16 to trigger any modulating signal. The necessity
for CD94 to be cross-linked with CD16 to inhibit CD16-mediated
production of the cytokine is similar to the CD94-mediated inhibition
of TNF- production via CD16 in NK cells.
Taken together, these results demonstrate that V9V2 T cells
behave like T cells through their response induced via TCR-CD3
stimulation but also behave like NK cells through their response
induced via CD16 and their regulation via CD94. This double
characteristic strongly suggests that these cells are not a redundant
population of T lymphocytes but represent an important class of
cells in the immune response that must play a key role in protection
against invasion by pathogens.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Virginie Lafont, Institut National de la Santé et de la Recherche Médicale, Unité 431, Microbiologie et Pathologie Cellulaire Infectieuse, Université Montpellier II, Place Eugène Bataillon, cc 100, 34095 Montpellier cedex 05, France. E-mail address: vlafont{at}crit.univ-montp2.fr
3 Abbreviations used in this paper: IPP, isopentenylpyrophosphate; ERK, extracellular signal-regulated kinase; MAPK, mitogen activated protein kinase; ATF, activating transcription factor; MEK, MAPK kinase; GamIg, goat anti-mouse Ig.
Received for publication December 21, 2000. Accepted for publication April 11, 2001.
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