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T Cells Stimulated with Soluble or Cellular Antigens1
Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Purpan, Toulouse, France
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Abstract |
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T, and NK lymphocytes establish immunological
synapses (IS) with their targets to enable recognition. Transfer of
target cell-derived Ags together with proximal molecules onto the
effector cell appears also to occur through synapses. Little is known
about the molecular basis of this transfer, but it is assumed to result
from Ag receptor internalization. Because human 
T cells
recognize soluble nonpeptidic phosphoantigens as well as tumor cells
such as Daudi, it is unknown whether they establish IS with, and
extract molecules from, target cells. Using flow cytometry and confocal
microscopy, we show in this work that Ag-stimulated human V9/V2 T
cells conjugate to, and perform molecular transfer from, various tumor
cell targets. The molecular transfer appears to be linked to IS
establishment, evolves in a dose-dependent manner in the presence of
either soluble or cellular Ag, and requires TCR ligation, Src
family kinase signaling, and participation of the actin cytoskeleton.
Although CD45 exclusion characterized the IS performed by T
cells, no obvious capping of the TCR was detected. The synaptic
transfer mediated by T cells involved target molecules unrelated
to the cognate Ag and occurred independently of MHC class I expression
by target cells. From these observations, we conclude thatm despite the
particular features of T cell activation, both synapse formation
and molecular transfer of determinants belonging to target cell
characterize T cell recognition of Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T, and NK cells.
Whether or not T cells share these properties is unknown. Around
1–5% of lymphocytes in peripheral blood of healthy human adults
consist in T cells, most of which express TRGV2/TRDV2-encoded
TCR (referred to here as 92 lymphocytes) (see Ref.
19 for recent review). The reactivity of this subset is
mostly MHC unrestricted (20, 21, 22, 23) and is directed toward a
broad spectrum of Ags comprising, on the one hand, soluble nonpeptidic
ligands and, on the other hand, activated, virally infected, or cancer
cells such as the 2-microglobulin
(2m)- Burkitt lymphoma
Daudi (24, 25, 26, 27, 28). Surface receptors for MHC class I (NK cell
MHC receptor (NMR)) expressed by most T cells regulate the
recognition process (29, 30, 31, 32).
Although structurally diverse, the nonpeptide Ags activating 92
cells have low m.w. and hydrophilic structures. These comprise either
natural or synthetic pyrophosphoesters referred to as phosphoantigens
(33, 34), therapeutic aminobisphosphonates
(35), and natural or synthetic alkylamines
(36). Natural phosphoantigens such as 3-formyl-1-butyl
pyrophosphate (3fbPP) were isolated from various bacteria including
Mycobacterium tuberculosis (37) and
Escherichia coli (38). Among other powerful
phosphoantigens, isopentenyl pyrophosphate (IPP) is a ubiquitous
metabolite (39), and the synthetic agonist bromohydrin
pyrophosphate (BrHPP) was recently produced in our laboratory
(40). The phosphoantigen receptor on T cells was
unambiguously demonstrated as the V9/V2 TCR (41, 42), but the molecular basis of this recognition remains
unclear. Presumably, the V9/V2 TCR might accommodate distinct
phosphoantigens thanks to the direct binding of phosphate groups to the
positive charges of adequately exposed germline residues at the surface
of the Ag-binding region (42, 43, 44). Activation of 92
T cells also requires cell-to-cell contact but not MHC class I
expression, and thus might involve presentation by an
as-yet-unidentified molecule (22, 23). An attractive
candidate in this respect is the uncharacterized phosphatase
activity involved in activation by phosphoantigens
(45). In marked contrast with T cells,
T cell activation by phosphoantigens does not result in TCR
down-modulation (46, 47), which questions the ability of
the T cell to mediate synaptic capture of target cell Ags. We
sought to investigate this point using T cells stimulated with
either soluble or cellular Ags. This paper reports flow cytometry and
confocal microscopy data evidencing synaptic transfer by Ag-stimulated
92 T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BrHPP and 3fbPP were synthesized in our laboratory as previously
described (37, 40). Synthetic IPP was obtained from
Sigma-Aldrich (St. Louis, MO). Mouse mAb against human V9 TCR, human
IgM (clone AF6), and secondary goat anti-mouse Ab
(F(ab')2) conjugated to rhodamine were from
Immunotech (Marseille, France). Mouse mAb 10G10 against human
CD45 was a kind gift of P. Valitutti (Institut National de la
Santé et de la Recherche Médicale, Toulouse, France).
Cell culture
PBL from healthy donors were separated by density centrifugation on Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden), washed three times, and then resuspended at 106/ml in RPMI complete culture medium (RPMI 1640 with Glutamax-I (Life Technologies, Paisley, U.K.), supplemented with 10% FCS, 25 mM HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 1 mM sodium pyruvate).
Polyclonal 92 T cell lines were specifically raised by incubating
PBL (106/ml) in culture medium with 3fbPP (10 nM)
plus 100 U/ml IL-2 (Sanofi-Synthélabo, Labège, France)
during 20 days. The expansion of 92 T cells was followed by
cytometric analysis, and only cultures showing >95% TCR
V2-positive cells were used for subsequent experiments.
The THP-1 cell line was cultivated in RPMI complete culture medium plus 50 µM 2-ME. Raji, Daudi, OCI-Ly8, VAL, and C1R cell lines were grown in complete RPMI culture medium.
TNF- release assay
TNF- release by T cell lines was measured using a
bioassay described elsewhere (40).
Analysis of membrane capture by flow cytometry
Cells were stained with green lipophilic dye PKH67
(Sigma-Aldrich) or orange chloromethylbenzoylaminotetramethyl-rhodamine
(CMTMR; Molecular Probes, Eugene, OR) according to the manufacturer’s
instructions. PKH-labeled target cells were cocultured at
various ratio (1:1 when unspecified) with CMTMR-labeled T cells in
96-well U-bottom tissue culture plates at a final concentration of
4 x 105 cells in 100 µl with the
indicated concentration of phosphoantigen. Cells were centrifuged for 1
min at 700 rpm to promote cell contact and incubated for 1 h at
37°C. Cells were then washed twice with PBS containing 0.5 mM EDTA
and analyzed by flow cytometry. When specified, T cells were
pretreated with cytochalasin D (CytD), 10-6 M
(Calbiochem, Merck-Eurolab, Fontenay-sous-Bois, France) for 1 h at
37°C or with 20 µM pyrophosphate (PP)2 (Sigma-Aldrich) for 15 min
at 37°C before addition of target cells, and then cocultured with
these drugs.
Analysis of synaptic transfer or IS by confocal microscopy
Cells were stained as described above. Equal numbers of T
cells and target cells (usually 2 x 105)
were cocultured after centrifugation (1 min at 700 rpm) in 96-well
U-bottom tissue culture plates for 1 h at 37°C (synaptic
transfer experiments) or for 15 min (IS analysis) in specified
conditions. Cells were then gently resuspended and plated on
poly-L-lysine (Sigma-Aldrich)-coated slides for 5 min at
37°C. After fixation with PBS containing 4% paraformaldehyde, cells
were washed, blocked in PBS supplemented with 1% BSA (Sigma-Aldrich),
and processed for immunofluorescence. Primary Abs were added in PBS/1%
BSA for 1 h at room temperature and then washed extensively.
Secondary Ab were added in PBS/1% BSA for 30 min, washed extensively
in PBS, and then mounted in PBS containing 90% glycerol and 2%
1–4-diazabicyclo (2.2.2) octane (Sigma-Aldrich). Samples were examined
using a Zeiss LSM 410 confocal microscope (Zeiss, Jena, Germany). The
presented pictures were representative of results from at least three
different experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells actively capture membrane
fragments from accessory THP-1 cells
It is well known that, while T cells can be stimulated with
soluble Ags, the presence of accessory cells, including the T
cells themselves, is required. The myelomonocytic THP-1 cell line
exerts such accessory function for the response of polyclonal 92
T cells to nonpeptide Ags (48). Because recognition of
target cell by T, B, and NK cells involved the capture of target
cell molecules, we determined whether such a molecular transfer
occurred in conditions where THP-1 cells were cocultured with T
lymphocytes activated by the soluble Ag BrHPP. To unambiguously
identify each cell type, THP-1 were stained green with the lipophilic
dye PKH67, which inserts in cell membrane, while T cells were
stained orange with the cytoplasmic dye CMTMR. The PKH67 fluorescence
of gated live cells allowed us to discriminate between the
PKH67bright population corresponding to THP-1
cells and the PKH67low population corresponding
to T lymphocytes (Fig. 1
a). After 1 h of
coculture, the MFIPKH67 of the T cells
cultured with THP-1 cells alone (mean fluorescence intensity (MFI)
= 11 measured on gated CMTMR-positive T cells) reproducibly increased
when BrHPP was added to the coculture (MFI = 18), indicating the
capture of THP-1-derived membrane lipids by activated T cells.
The MFIPKH67 of T cells dropped to 6 in
cocultures where both BrHPP and the Src family kinase inhibitor PP2
were added (Fig. 1a). The capture of THP-1 membrane fragment
by unstimulated T cells was weak but reproducible and was
lowered by the addition of PP2 to the coculture (see Fig. 4b). Increasing the ratio of THP-1: T cells also
increased the amount of target cell lipids acquired by BrHPP-stimulated
T cells; the MFIPKH67 reached 44 at the
highest E:T ratio tested (Fig. 1b). As expected from the
weak reactivity of unstimulated T cells to THP-1 cells alone,
this capture measured in these conditions was marginally affected by
the above cell ratio. When a large excess of THP-1 cells was used the
effect of PP2 on membrane capture by activated T cells was
slightly less marked, possibly reflecting consumption by THP-1 cells
(Fig. 1b). Nevertheless, PP2 reduced membrane capture to
levels observed with unstimulated T cells. Altogether, these
data indicated that, like other lymphocyte populations, T cells
activated by soluble phosphoantigens capture membrane fragments of
accessory THP-1 cells.
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T cells
T cells respond to different concentrations of the soluble
phosphoantigens IPP, BrHPP, and 3fbPP as well as to the
2m-deficient Burkitt’s lymphoma cell line
Daudi (24, 49). To probe the qualitative and quantitative
relationship between antigenic stimulation of T cells and
membrane capture further, we tested various doses of these Ags in
cocultures of T cells and THP-1 cells as above. The transfer of
THP-1 membrane fragments onto T cells augmented with increasing
concentrations of BrHPP, IPP, or 3fbPP in a dose-dependent
manner. Half-maximal capture correlated well to the known
biological activity of these compounds, as assessed in functional
assays (33, 37, 39). Thus, BrHPP or 3fbPP induced
half-maximal THP-1 membrane capture at roughly
103-fold lower concentrations than IPP (i.e., 200
or 50 nM vs 200 µM, respectively; Fig. 2a).
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T cells and Daudi
tumor cells were incubated separately and mixed immediately before flow
cytometric analysis (duration of cell contact: 0 min; Fig. 2
b, 
). By comparison, after 1 h of coculture, the
MFIPKH67 of T cells was markedly
increased. As observed above with soluble Ags an increase in Ag
density, as obtained in increasing the Daudi: T cell ratio,
resulted in a more efficient transfer of Daudi determinants on T
cells (Fig. 2b). Interestingly, at the same target
cell: T cell ratio, the capture of THP-1 membrane fragment in the
presence of high phosphoantigen concentrations was equivalent to the
capture of Daudi cell membrane alone. Thus, regardless of the stimulus
(soluble or cellular), T cell activation resulted in a marked
capture of membrane fragments from cells to which they were
conjugated.
T cells acquire molecules from target cells after synapse
formation
To visualize the process of molecular transfer between T
cells and either THP-1 or Daudi cells, we repeated the experiments
presented above and analyzed them by confocal microscopy. A hallmark of
IS performed by T and B lymphocytes in the presence of Ag is the
exclusion of CD45 from the area of contact with target cells. Thus, we
used an anti-CD45 mAb to analyze conjugates between T and
THP-1 cells in the presence or absence of BrHPP. As shown in Fig. 3, the uniform CD45 staining observed on
T cells conjugated to THP-1 in the absence of BrHPP was replaced
by a discontinuous staining, CD45 being largely excluded from the
contact area when BrHPP was added. A comparable CD45 exclusion was
observed in conjugates formed between T cells and Daudi (Fig. 3, a and b). Thus, CD45 exclusion applies to
T cell synapse as well.
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TCR at the
contact area (Fig. 3, a and b). When
PKH67-labeled THP-1 cells were cocultured with CMTMR-labeled T
cells, a clear PKH67 staining was observed on T cells when BrHPP was
present but not in its absence. Interestingly, the presence of PKH67 on
T cells was the most noticeable at the level of the IS (Fig. 3a). Captured membrane patches spread out of the contact
area at the T cell surface, localizing at the membrane and
submembrane levels of the IS (Fig. 3c, arrows). In the
presence of PP2 or CytD, BrHPP-induced membrane capture was markedly
inhibited (Fig. 3c). Conjugates of T cells and THP-1
cells were observed in each experiment, although they were more
frequent with BrHPP. In contrast, PP2 and CytD significantly prevented
conjugate formation (Table I). In
agreement with the former cytometry experiments, membrane capture by
unstimulated T cells was very limited, whereas large green patches
derived from THP-1 membranes were observed at the surface of
BrHPP-stimulated T cells. Similarly conjugated T cells
were less abundant in cocultures containing BrHPP and PP2 or BrHPP and
CytD (Table I). In neither condition did T cells without
synaptic engagement harbor any green patch of THP-1 membrane (data not
shown). The T cells conjugated to Daudi cells also led to a
synaptic transfer of PKH67 dye, which increased in the presence of
BrHPP and decreased in the presence of PP2 or CytD as found above with
THP-1 cells (Fig. 3d). To document the molecular transfer of
membrane markers from Daudi to T cells further, the conjugates
were analyzed for surface expression of IgM. For comparison, these
cocultures were also done in medium containing either PP2 or CytD. As
shown in Fig. 3e, a clear transfer of IgM from Daudi to
T cell surface was observed except when PP2 or CytD was added
(Fig. 3e).
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T cells
establish an IS with target or accessory cells through
which theycapture target cell surface molecules including lipids and
proteins such as IgM. This process was referred to as synaptic
transfer.
Phosphoantigen inhibitors reduced synaptic transfer by T
cells
We had previously shown that phosphoantigen recognition
involves dephosphorylation by an as-yet-unidentified phosphatase
(45). Because the efficiency of synaptic
transfer by T lymphocytes correlated with Ag
potency, we tested the effect of phosphatase inhibitors on the capture
process. At millimolar (nontoxic; data not shown) concentrations,
levamisole and inorganic PP (PPi) severely inhibited the release of
TNF- by BrHPP-stimulated T cells but did not affect mitogenic
(PHA) activation of T cells (Fig. 4a). When tested in membrane
capture assays, levamisole and PPi markedly inhibited synaptic
transfer. In addition, a strong inhibition of PKH67 transfer was also
obtained when the Src family kinase inhibitor PP2 or the
cytoskeleton remodeling inhibitor CytD were used (Fig. 4b). Altogether, the pharmacological control of membrane
capture indicates that synaptic transfer requires the selective
recognition by the TCR of phosphoantigens and subsequent T cell
activation involving Src family kinase activity and remodeling of the
actin cytoskeleton.
Activation-induced synaptic transfer from Daudi cells
As indicated by the above results, T cells readily capture
membrane fragments from the Daudi lymphoma in the absence of
phosphoantigen added. Addition of PP2 or BrHPP, respectively, inhibited
and enhanced the synaptic transfer by T cells in 1 h of
coculture with Daudi (Fig. 5a). As the
2m-deficient Burkitt’s lymphoma Daudi does
not express MHC class I molecules, its recognition by T cells may be
controlled not only by the 92 TCR but also by NK receptors for
MHC class I expressed at the T cell surface
(29, 30, 31, 32). Therefore, we compared the extent of synaptic
transfer exerted by T cells coincubated either with Daudi or
with 2m transfectants of Daudi cells that
express surface MHC class I molecules (51). The MHC class
I molecule expression was verified before each transfer assay by
immunofluorescence using W6/32 mAb (data not shown). When cocultured
for 1 h with 2m+
Daudi, PKH67 was transferred onto T cells in quantitatively
similar extent as with untransfected Daudi. This active capture process
was again reduced by PP2 and increased by BrHPP (Fig. 5b).
Therefore, surface expression of MHC class I molecules by engaged
targets did not affect synaptic transfer by T cells, which
appears to be mediated mainly by the 92 TCR.
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The above data suggested that activated T cells capture
membrane fragments from tumor cells regardless of MHC class I
expression. To strengthen this observation, we tested other tumoral
cell lines that elicit different biological responses from T
cells. We used the MHC class I-negative K562 cell line, which is lysed
by T cells in an NK-like TCR-independent fashion
(52). The non-Hodgkin lymphomas VAL and OCI-Ly8 were
chosen as efficiently killed targets by T cells
(28), and Raji (29) and C1R (data
not shown) were chosen as nonlysed cell lines. The T cell
responses to those tumor cell lines are summarized in Table II. Only a marginal synaptic transfer
from C1R occurred, which was PP2 resistant and increased by BrHPP. All
other tumor cells induced a strong transfer onto T cells, which
was moderately increased in the presence of BrHPP. In addition, the
membrane capture observed for these tumor cell lines was only slightly
inhibited by PP2 (Fig. 5c). Thus, in agreement with the
former results, T cells captured membrane fragments from most of
their conjugated tumor targets, regardless of whether these latter
expressed MHC class I molecules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells (5, 6, 9), B cells (7, 53), and NK cells (15, 16, 18)4 by their respective Ags involves the
stripping of target cell membrane fragments that convey cell surface
proteins including Ags. The present study demonstrates for the first
time that, once activated by either soluble or cellular Ags, human
T cells also capture target cell molecules. Because cell-to-cell
contact is required for T cell activation by soluble nonpeptide
Ags, we first used the THP-1 myelomonocytic cell line (providing a
convenient accessory cell) in the presence of phosphoantigens
(48, 54) as a T cell stimulus. Activation by
phosphoantigens triggered an active transfer of THP-1 membrane patches
by the T cell effectors. We also analyzed this process in a
second model involving the innate T cell reactivity for tumor
cells such as Daudi, in the absence of exogenously supplied
phosphoantigens. In this case as well, T cells conjugated to
Daudi acquired molecules from the target cell surface.
ISs are tight contact zones that reactive lymphocytes establish with
their cell targets. In this contact area, some molecules are enriched
and others are excluded. For instance, exclusion of CD45 from the
cell-cell contact region is a hallmark of IS (4, 50).
Based on this criterion, T cells seem to establish authentic IS
with their conjugated targets. The building of IS also relies upon the
highly dynamic remodeling of the actin cytoskeleton, mediated by inter
alia ATP, Ca2+, and protein kinase C
(2, 17). Consequently, inhibitors targeting the
cytoskeleton such as CytD profoundly affect the ability of lymphoid
cells to set ISs, to transduce activation signals, and to internalize
ligands from their surface receptors (10, 55). In this
respect, the synaptic transfer by T cells was crucially
sensitive to inhibitors of actin cytoskeleton. This is very reminiscent
of TCR- and CD28-mediated ligand uptake as performed by T cells
(10) or NMR-mediated capture by NK
cells.4 Because an IS may appear in the absence
of Ag (56), we wanted to ascertain whether cognate
phosphoantigen recognition was necessary in our model. T cell
activation by BrHPP involves hydrolysis of the PP group by an
unidentified phosphatase and release of the inactive monophosphate
byproduct (45). We thus used levamisole as
phosphohydrolase inhibitor (57) and PPi. Presumably by
competing with PP moiety of phosphoantigens, PPi was found to
specifically inhibit phosphoantigen-induced activation. Both molecules
inhibited phosphoantigen activation and impaired BrHPP-induced membrane
capture. Hence, the synaptic transfers was subordinated to the Ag
recognition mechanism, including phosphoantigen hydrolysis.
In line with previous studies (9),4
the potent inhibition caused by PP2 demonstrated that synaptic transfer
required Src family kinase activity. For T cells, the level of
TCR occupancy by Ag is usually translated into an Ag dose-dependent
activation of protein tyrosine kinases (58); therefore, we
assume that Src family kinase activation was triggered by TCR
engagement. The occupancy of TCR by phosphoantigens cannot be
monitored as for T cells (46, 47). Nevertheless,
synaptic transfer strictly followed the dose-dependent T cell
activation by either soluble or cell Ags, and was markedly inhibited by
inhibitors of phosphoantigen recognition. Furthermore, the experimental
conditions allowing synaptic transfer to occur had previously been
shown to induce a sustained signaling in polyclonal T cell
(46). Altogether, these results allow us to conclude that
T cells do not apparently differ from other lymphocyte
populations in their manner to interact with targets and to capture
surface molecules from them (4). Thus, in T
cell-target cell conjugates, TCR triggering led T cells to
establish an IS, to reorganize their actin cytoskeleton, and to
initiate sustained intracellular signaling, enabling synaptic transfer
of target cell molecules onto the T cell.
However, the following lines of evidence seem to diverge from this
standard concept. Synapse formation by T cells is thought to
concentrate locally both TCR and signaling molecules at central
supramolecular activation clusters (1, 3, 59).
Although T cells apparently established typical IS with target
cells, no macroscopic TCR polarization at the IS was noticed.
However, this situation was previously reported, and either high
dissociation rates of cognate complexes or different kinetics of
synapse establishment and TCR polarization were proposed to explain the
absence of TCR enrichment in IS (50, 60). In the present
model, it is also possible that the phosphoantigen concentration or Ag
density at the tumor cell surface was high enough to trigger both
sustained signaling and synaptic transfer without the need for
enrichment of TCR. In the case of CD4+ or
CD8+ T cells, ligand uptake is thought to be the
consequence of TCR internalization (5, 7, 9).
However, recognition of potent phosphoantigen agonists by cell surface
TCR does not involve TCR down-modulation (46, 47). Therefore, T cell-mediated synaptic transfer
induced by antigenic stimulation is not the mere consequence of TCR
internalization.
The recognition of Daudi cells is mediated by the TCR
(24, 25, 26, 61). Accordingly, coculture of Daudi cells with
92 T cells led to a synaptic transfer that was slightly increased
by adding BrHPP to the coculture or was abrogated by T cell
treatment with PP2. As already reported for CTL (9)
and NK cells,4 the capture of target cell
membrane fragments by T cells occurred via the IS. Thus,
synaptic transfer is unrelated to the nature of the TCR ligand,
as soluble or cellular Ags activated a comparable membrane capture by
T cells.
Exposure of T cells to increasing numbers of Daudi targets
increased synaptic transfer correspondingly. This observation is
consistent with the so-called "kiss and run" phenomenon underlying
the serial target cell killing by CTL. During this process we observed
the capture of target cell markers such as B cell-derived Ig on the
T cell surface. This capture can be visualized as small IgM
aggregates distributed on the surface of T cells. The role
that IgM or other surface molecules may play once they are acquired by
T cells is elusive and requires further investigation.
Recognition of Daudi by T cells involves the 92 TCR
(26) but consequent activation is controlled by NMR, as
testified by the higher sensitivity of Daudi as compared with
Daudi-2m+ transfectants
to T cell-mediated lysis (29, 30, 31, 32). In contrast to
cytotoxicity assays, T cells captured membrane material from
Daudi and Daudi-2m+
tumor cells to similar extents. Therefore, the expression of MHC class
I molecules at the Daudi cell surface does not hamper the synaptic
transfer. A panel of tumor cell lines, selected according to
their MHC class I molecule expression and effector responses (Table II), reinforced theses observations. As for Daudi cells, synaptic
transfer occurred with these tumor cells regardless of their MHC class
I molecule expression. Furthermore, no correlation appeared between the
ability of those cell lines to be subject to synaptic transfer and
their ability to trigger cytotoxicity or amplification. This
dissociation from effector responses was already noticed using
perforin-deficient T cells (9) or B cell models
(7). So human T cells exerted synaptic transfer
from tumor cells, but without strong inhibitory effect delivered by
their NMR. This suggests that synaptic transfer is an early process
occurring while the T cell is scanning its conjugated cell surface.
Nevertheless, as shown with C1R cells, not all tumoral cell lines allow
this phenomenon. The difference with the highly controlled synaptic
transfer performed by NK cells (18)4
may be due to the dominant activatory signal delivered by TCR.
In addition, T cell response to lymphoma often involves CD28
coengagement (62, 63, 64), which also potentiates the synaptic
transfer (6). TCR-mediated or NK receptor-mediated
target cell recognition may lead to synaptic transfer. Phosphoantigen
addition frequently enhances the T cell responses to non-Hodgkin
lymphoma (28). Also, T cells exert quantitatively
similar synaptic transfer from TCR-dependent and -independent cell
targets Daudi and K562, respectively. Current studies in our laboratory
aim at delineating the respective contributions and outcomes of TCR and
NK receptor signals in this IS and the associated transfer.
By visualizing membrane bridges between CTL and target cell surface,
Stinchcombe et al. (11) proposed that the continuity of
fused membrane bilayers enabled the direct molecular diffusion from
target to effector cell at the level of the IS. Taken together, our
observations fully agreed with Stinchcombe’s model of membrane ripping
by nonselective lateral diffusion (11). Furthermore, this
process permits the transfer of target cell surface molecules such as
IgM in proper orientation. Synaptic transfer induced by activation of
T cells depended upon the highly active establishment of a
functional IS. By allowing the local formation of membrane
continuities, the synapse most likely enables lateral diffusion of
surface molecules originating from the target, regardless of TCR
internalization. However, the physiological role of this process still
remains unclear. By acquiring target cell-derived Ags, T cells
like T cells may become themselves subjected to CTL lysis or
fratricide killing (5). Hence, synaptic transfer could
favor the termination of immune responses via lymphocyte exhaustion
(6). Alternatively, a competition between reactive T cells
for Ag stripping from APC surfaces might drive affinity maturation of
the in vivo immune response (65). However, in the present
context this hypothesis seems unlikely because no biased selection of
lymphocytes bearing higher-affinity receptors was observed in response
to phosphoantigen stimulation evaluated by the size profiles of their
TCRV2 CDR3 (66). Future work will now focus on the
elucidation of the physiological role of synaptic transfer by T
cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Jacques Fournié, Institut National de la Santé et de la Recherche Médicale, BP3028 Centre Hospitalier Universitaire Purpan, 31024 Toulouse, France. E-mail address: fournie{at}toulouse.inserm.fr
3 Abbreviations used in this paper: IS, immunological synapse; PP, pyrophosphate; BrHPP, bromohydrin PP; CMTMR, chloromethylbenzoylaminotetramethyl-rhodamine; 3fbPP, 3-formyl-1-butyl-PP; IPP, isopentenyl PP; MFI, mean fluorescence intensity; NMR, NK cell MHC receptor; 2m, 2-microglobulin; CytD, cytochalasin D; PPi, inorganic PP.
Received for publication February 11, 2002. Accepted for publication April 8, 2002.
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)
inhibit BrHPP-induced but not PHA-induced (
, respectively)
) and 3fbPP (shaded circles).
Negative control (
