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Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, U277, Institut Pasteur; and
Laboratoire d’Immunologie Cellulaire et Institut National de la Santé et de la Recherche Médicale CJF9711, Hôpital de la Pitié-Salpêtrière, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the limited number of Ags on the surface of APC, the weak affinity of the TCR for its ligand, as well as the monomeric nature and the small size of the TCR, the T cell needs a specialized contact zone with the APC (termed immunological synapse) to be the site where the antigenic ligand will activate the T cell (3, 4). When signal-2 is absent, T cell stimulation requires a more potent and prolonged signal-1 (5). Thus, costimulatory molecules such as CD28/B7 apparently increase both the duration and the amplitude of the signals transduced through the TCR. Moreover, signal-2 seems to be involved in cell survival, probably through the up-regulation of survival factors such as Bcl-xL (6). It has been recently demonstrated that costimulatory molecules play a role in synapse formation by initiating the actin- and myosin-based transport of receptors and protein complexes toward the cell contact zone (7). The CD28 engagement also initiates the redistribution of cell surface lipidic microdomains ("rafts") to the contact site with the TCR (8). This phenomenon probably allows the amplification of signals transduced through the TCR via the segregation of relevant molecules.
These studies seem to imply that the two stimuli must be colocalized and given simultaneously for T cell stimulation to be optimal. For example, Liu and Janeway showed that clonal expansion of normal CD4+ T cells did not occur when the two signals were delivered by separate cells (9). In contrast, Ding and Shevach, in a similar model system but using APC expressing high levels of B7 molecules, demonstrated that naive CD4+ T cells can be activated as efficiently by costimulation in trans as by costimulation in cis (10). Previous studies of cloned T cell lines also suggested that the two signals could be delivered by separate cells (11, 12, 13, 14).
We have examined whether naive CD8+ T cells need simultaneous signals 1 and 2 to be optimally stimulated. We previously set up an in vitro activation system for naive CD8+ T cells freshly purified from TCR transgenic mice (15). In this system the cells were stimulated in vitro using recombinant MHC/peptide complexes (signal-1) in the presence or the absence of costimulatory anti-CD28 mAb (signal-2), both signals being immobilized on plastic, in the absence of APC. We used this flexible system to examine the effect of duration of each signal independently on the T cell activation as well as the effect of sequential vs simultaneous delivery of signal-2. We also assessed the importance of signal-1 strength on separate costimulation by using as signal-1 a superagonist peptide ligand complexed with the MHC molecules. Finally, we examined the precursor cell number for various timings and durations of signal-2. Our results indicate that separate and delayed costimulation can potentiate the signal-1 response and increase precursor number. These data support the idea that signal-1 stimulation may result in a transient "excited" state of CD8+ T cells, in which the cells remain susceptible to costimulation. Possible physiological relevance and in vivo implications are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR transgenic clone 4 mice, transgenic for a Kd-restricted TCR specific for the HA peptide (512-520) derived from the hemagglutinin protein of the influenza virus (16) were bred and kept at the Pasteur Institute (Paris, France) under pathogen-free conditions according to institutional guidelines.
Reagents and Abs
Carboxy-fluorescein diacetate succinimidyl ester (CFSE;2 Molecular Probes, Eugene, OR) was dissolved in DMSO at a concentration of 20 mM and stored at -20°C. Recombinant single-chain Kd (SC-Kd) was prepared and loaded with either HA (IYSTVASSL) or 6G (IYSTVGSSL) peptide as previously described (17). SF1-1.1.1 mAb (HB159) was obtained from American Type Culture Collection (Manassas, VA). Anti-CD28 mAb, biotinylated SF1-1.1.1 mAb, anti-CD8 mAb, and streptavidin-PE were purchased from PharMingen (San Diego, CA). Magnetic microbeads coupled to goat anti-rat IgG were purchased from Miltenyi Biotec (Auburn, CA).
Purification of CD8+ splenocytes
CD8+ T cells were purified by positive selection from total splenocytes of Tg mice as described previously (15). Briefly, 108 red cell-depleted splenocytes were treated with purified anti-CD8 mAb for 35 min at 4°C and washed. Magnetic microbeads coupled to goat anti-rat IgG were added for 15 min at 4°C. The splenocytes were washed and separated by chromatography on a column attached to a magnet. The purified population routinely consisted of >98.5% CD8+ T cells, as revealed by FACS analysis.
In vitro transfer experiments and T cell proliferation assay
Recombinant single-chain MHC molecules loaded with the HA peptide (SC-Kd/HA) was dimerized with Kd-specific Ab SF1-1.1.1 (signal-1) in stoichiometric amounts for 30 min at room temperature. The complexes were immobilized onto flat-bottom 96-well plastic plates for 16 h at 4°C (15). Immobilization was followed by three washes with PBS. Purified CD8+ T cells were cultured for various times in RPMI 1640 medium supplemented with 10% heat-inactivated FCS in the coated wells. The plates were centrifuged immediately after set-up to synchronize T cell activation. The cells were then transferred either to uncoated culture plates to terminate the activation or to culture plates coated with 20 µg/ml anti-CD28 mAb to deliver signal-2 until a total time of 48 h was reached. In control experiments using photoreactive plates (Universal-BIND, Costar, Cambridge, MA), the SC-Kd/HA-coated plates were UV light irradiated for 2 min in an auto-cross-link mode in a Stratalinker UV cross-linker (Stratagene, La Jolla, CA). In the experiments using strong signal-1, the HA peptide was replaced in the complexes by altered peptide 6G (SC-Kd/6G). In certain experiments the duration of signal-2 was limited to 60 min before the cells were transferred again to uncoated wells until reaching 48 h. In time lag experiments, the cells were transferred after contact with signal-1 to uncoated wells for 30 or 120 min at 37°C, then transferred again to anti-CD28-coated wells until reaching 48 h. In some experiments both SC-Kd complexes and anti-CD28 mAbs were immobilized together overnight at 4°C. In any case, [3H]thymidine was added to the cells after 32 h of culture, and incorporation was measured after 16 h of labeling.
CFSE labeling
Immediately after purification, CD8+ T cells were washed with ice-cold PBS and resuspended at 107 cells/ml in PBS. Cells were incubated at room temperature for 10 min with CFSE at a final concentration of 10 µM. After labeling, 1 vol of FCS was added to the cell suspension, and the cells were centrifuged and washed three times with ice-cold PBS before being resuspended in PBS.
Cytometric analysis
Cell division analysis was performed on CFSE-labeled CD8+ T cells (3 x 105/well) by flow cytometry. Gates to exclude nonviable cells were determined using propidium iodide staining (Sigma). Data for live cells were acquired in a FACScan and analyzed using CellQuest software (Becton Dickinson, San Jose, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the influence of sequential contact with signals 1
and 2 on CD8+ T cell activation, we performed in
vitro transfer experiments in which the cells were transferred at
various time into empty wells to disrupt contact between the signals
and the cells (Fig. 1
). After 20, 60, or
180 min of contact with 800 ng/ml of SC-Kd/HA (signal-1), the T
cells proliferated, as measured by
[3H]thymidine incorporation, and the level of
proliferation increased with the time of contact, indicating that the
contact with signal-1 was really terminated at the time indicated (Fig. 1, signal-1 only). However, when the cells were transferred into wells
coated with 20 µg/ml of anti-CD28 mAb (signal-2) instead of empty
wells for 48 h, the proliferative response clearly increased for
each time of contact with signal-1 (Fig. 1, +signal-2). The maximum
amount of proliferative response was obtained when signal-1 was
delivered alone for at least 60 min, followed by signal-2. As expected
from previous experiments (15), the proliferative response
was peptide specific, because no response was observed when complexes
bearing the irrelevant peptide CW3 were used instead of SC-Kd/HA
even in the concomitant presence of signal-2 (Fig. 1, irrelevant
+signal-2). The cells responded to mitogenic stimulation (Fig. 1, conA)
and did not proliferate in the presence of signal-2 alone (Fig. 1, signal-2 only). This result demonstrates that potentiation of signal-1
by signal-2 was observed when the two signals were delivered
sequentially, with signal-1 of short duration.
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). A proliferative response could be
observed when signal-1 alone was added to the cells in a soluble form
for 48 h, which was lower than that obtained with the immobilized
signal-1 for the same amount of time as well as that obtained when the
cells were stimulated with the two immobilized signals together.
However, no increase in the response was detected either when
immobilized signal-2 was delivered to cells that had been previously in
contact with soluble signal-1 or when the cells were pretreated with
immobilized signal-2 before immobilized signal 1.
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Finally, to confirm that potentiation could be observed without
possible carryover of signal-1, we examined the proliferation response
to signal-1 alone or followed by signal-2 (with or without delay, see
below) using a signal-1 covalently bound to plastic plates through UV
light irradiation. The Sc-Kd/HA complexes were dimerized using a
biotinylated Ab that recognizes the 
3 domain of the MHC molecule.
Upon streptavidin-PE staining, we failed to detect by FACS analysis
significant carryover of the complexes along with the cells (data not
shown). Using both classical and photoreactive plates in parallel
experiments, we performed transfer experiments after various contact
times with signal-1. The cells were transferred onto plates harboring
either no signal-2 or immobilized signal-2, with or without a time lag
between the delivery of signals 1 and 2 (see below). We observed no
significant difference in proliferation levels or in potentiation
between conventional and UV light-treated plates (data not shown).
Thus, carryover is not responsible for the potentiation we observed
upon separate costimulation of CD8+ T cell activation.
Effect of a time lag between the delivery of signals 1 and 2
To test the effect of a delay in costimulation delivery, we
designed an experiment in which a time lag between the two signals was
allowed (Fig. 1
). After CD8+ T cells were
stimulated with immobilized signal-1 for various times, they were
transferred to empty wells for 30–120 min before being transferred
again to wells coated with signal-2 (Fig. 1, 30-min delay and 120-min
delay). Although a time lag of 30 min did not significantly decrease
the potentiation effect of signal-2 (Fig. 1, 30-min delay), a longer
time lag was more detrimental, but did not obliterate the effect
completely (Fig. 1, 120-min delay). Thus, signal-1-stimulated cells
remained susceptible to separate and delayed costimulation for at least
2 h. It is interesting to note that the time lag effect was not
influenced by the signal-1 duration. Indeed, a 2-fold increase in T
cell proliferation was observed when signal-2 was added 30 min after
contact for 20, 60, or 180 min with signal-1 (Fig. 1, compare 30-min
delay for 20, 60, and 180 min of signal-1 duration), falling to a
1.5-fold increase for a longer delay at each time point (Fig. 1, compare 120-min delay for 20, 60, and 180 min of signal-1
duration).
Proliferative response of naive CD8+ T cells to sequential contact with strong signals 1 and 2
To examine the effect of signal-1 strength on costimulation
potentiation, we used an altered peptide ligand bearing a A to G
mutation at position 6 of the parental HA peptide to stimulate
CD8+ T cells. This altered peptide ligand was
shown to activate CD8+ T cells when loaded onto
APC at concentrations, on the average, 25 times lower than that needed
by the parental HA peptide and was therefore identified as a strong
peptide agonist (C. Cambouris, unpublished observations). A strong
peptide agonist (or superagonist) is defined as a mutant peptide that
has a greater activation capability than its parental counterpart
(18). This strong agonist was used to form SC-Kd
complexes that were immobilized on plastic and used as signal-1. Like
the SC-Kd/HA complexes under the same conditions, the immobilized
SC-Kd/6G complexes could activate CD8+ T
cells in a peptide-specific, dose-dependent manner (Fig. 2). At high concentration, the extent of
proliferation was equivalent for both SC-Kd/HA and SC-Kd/6G
complexes (Fig. 2, lanes SC-Kd/HA and SC-Kd/6G at 8000
ng/ml). However, at a low concentration of complexes, the T cells
proliferated more extensively when stimulated with the SC-Kd/6G
complexes (Fig. 2, compare lanes SC-Kd/HA and SC-Kd/6G at 80
and 800 ng/ml). Indeed, the SC-Kd/HA concentration required to
reach half-maximal T cell proliferation was 700 ng/ml, while that of
SC-Kd/6G complexes was 200 ng/ml. This result confirmed the strong
agonist nature of the 6G peptide observed with the experiment
using APC.
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(Fig. 3). As with the parental signal-1, after
20, 60, or 180 min of contact with the SC-Kd/6G complexes, the T
cells proliferated (Fig. 3, signal-1 only). However, 180 min of contact
with the strong signal-1 alone was sufficient to achieve the maximum
proliferative response. Such a level of proliferation was similar to
that of CD8+ T cells that have been stimulated
simultaneously with SC-Kd/HA complexes and signal-2 for 48 h
(see Table I) or SC-Kd/6G complexes and signal-2 for 48 h
(not shown). After 180 min of contact, the proliferative response to
the parental signal-1 alone (SC-Kd/HA) reached only 50% of its
maximum in our experimental conditions (compare Figs. 1 and 3, signal-1
only). When signal-2 was delivered after various times of contact with
the strong signal-1, an increase in proliferation was observed (Fig. 3, +signal-2). The proliferative response was maximum after only 20 min of
contact with strong signal-1 followed by signal-2. In the previous
experiment, 60 min of parental signal-1 followed by signal-2 were
required for maximal stimulation (compare Figs. 3 and 1, +signal-2 at
20 and 60 min of signal-1 duration, respectively). The maximum level of
signal-2 potentiation was similar whether the signal-1 used to
stimulate CD8+ T cells was the parental
SC-Kd/HA or strong SC-Kd/6G signal (compare Figs. 1 and 3,
respectively). However, in contrast to the situation with parental
signal-1, proliferation in response to strong signal-1 for 180 min
could not be increased by subsequent contact with signal 2.
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The effect of a delay in the delivery of signal-2 after contact
with the strong signal-1 was tested in an experiment identical with
that performed using parental signal-1 (Fig. 3). We observed that
regardless of the duration of contact with strong signal-1, a time lag
of up to 120 min between strong signals 1 and 2 did not affect the
increase in proliferative response (Fig. 3, 120-min delay). Thus,
increasing the strength of signal-1 allowed a longer delay between the
two signals, while increasing only the duration of parental signal-1
did not (compare in Figs. 3 and 1, 30-min delay and 120-min delay at
20, 60, and 180 min of signal 1).
Effect of contact time with signal-2 on the proliferative response
To investigate whether potentiation was dependent of costimulation
duration, we tested the effects of various signal-2 durations after a
60-min stimulation with either parental or strong signal-1 (Fig. 4). When signal-2 was given for 60 min
immediately after SC-Kd/HA or SC-Kd/6G signal, an increase in
the proliferative response was observed (Fig. 4, +signal-2 (60 min)).
However, potentiation was more pronounced when signal-2 was delivered
for 48 h with either parental or strong signal (Fig. 4, +signal-2
(48 h)). Interestingly, the potentiation level was similar whether
signal-2 was given for 48 h after 60 min of signal-1 or signal-1
and signal-2 delivered altogether for 60 min (Fig. 4, signal-1+2 (60
min)). Prolongation of signal-2 for 48 h after the two signals
were delivered together for 60 min did not increase the proliferative
response (not shown). Thus, the effect of cosignal duration is
independent of the strength of signal-1. Moreover, potentiation of
signal-1 by a long separate signal-2 is equivalent in terms of
proliferative response to that of both signals given together.
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To test the effect of potentiation on cell division, we used
CFSE-labeled CD8+ T cells for in vitro transfer
experiments followed by FACS analysis. Upon cell division, CFSE
segregates between daughter cells so that the fluorescence intensity of
the cells is divided in half with each successive generation
(19). The populations of cells for each cell division are
visualized as distinct peaks, as shown in Fig. 5. The delivery to the
CD8+ T cells of either signal-1 alone for 20 min
(Fig. 5A), signal-1 followed by a long (48-h) or a short
(60-min) signal-2 (Fig. 5, B and C,
respectively), or signal-1 together with signal-2 for 20 min (Fig. 5D) resulted in up to four cell divisions. This result
demonstrated that the addition of signal-2 did not increase the maximum
number of cell divisions that the T cells undergo upon signal-1
stimulation and contrasts with the increase in thymidine incorporation
observed in the proliferation experiments. Increasing the duration of
signal-1 to 60 or 180 min in the presence or the absence of concomitant
or sequential signal-2 did not result in more cell divisions (data not
shown). As previously reported by others (20), about 25%
of the cells do not divide after contact with signal-1. In fact, these
cells did not express the surface cell activation marker CD69 (data not
shown). It is interesting to note that the timing of addition of
signal-2 did not alter the percentage of cells that never entered cell
division. However, we observed a clear increase in division peak 3 when
signal-2 was present and regardless of the time of signal-2 addition
(Fig. 5, compare A to B–D).
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). This analysis revealed that the
number of precursor cells undergoing zero, one, or two cell divisions
was unaffected by the addition of signal-2 (Fig. 6, signal-1 only,
+signal-2 (60 min), and signal-1+2 for zero, one, and two cell
divisions). However, twice as many precursor cells underwent three
divisions or more when contact with signal-1 was followed by contact
with signal-2 (Fig. 6, +signal-2 for three cell divisions) as well as
when both signals were added together (Fig. 6, signal-1+2 for three
cell divisions). Even 60 min of contact with signal 2 after
signal-1 caused 50% more cells to undergo three divisions (Fig. 6, +signal-2 (60 min) for three cell divisions). Therefore, the length of
time that signal-2 was delivered seems to have an influence on the
number of precursors present in division peak 3. These results
demonstrate that when signal-1 is short, signal-2 causes more precursor
cells to undergo multiple cell divisions regardless of the timing of
costimulus addition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Taken together, these results demonstrate that colocalized and concomitant signals 1 and 2 are not essential for optimal stimulation, and that costimulation in trans occurs during the activation of naive CD8+ T cell in vitro. Interestingly, increasing the strength rather than the duration of signal-1 allowed the delay in the delivery between the two signals to be extended, suggesting that delayed costimulation may be modulated by signal-1 strength.
In earlier studies CD28-mediated T cell costimulation was shown to sustain the late proliferative response as well as enhance long-term cell survival (6). We, on the other hand, demonstrate here that even though costimulation is not essential for early proliferative responses, it is able to potentiate the signal-1 effect early in the stimulation process. In fact, similar potentiation of signal-1 by signal-2 was observed in any conditions when transfer experiments identical with those described above were tested after 72 h rather than 48 h (data not shown). Indeed, most of what is known about the role of CD28 molecules in T cell activation comes from studies in which both signals are delivered separately or together, but signal-1 is not removed. It is possible that elimination of signal-1 may be important for signal-2 potentiation.
Previous studies using a cytotoxic CD8+ clone demonstrated that costimulation could be provided separately and independently of signal-1 (11). Interestingly, activation without costimulation in this system led to TCR-mediated cytotoxicity in the absence of IL-2 production. In contrast, in our system using naive CD8+ T cells, costimulation was not required for TCR-dependent effector functions (15).
"Trans-costimulation" has also been described for the CD4+ T cells. In the case of CD4+ T cell clones, costimulation could be provided by separate irradiated bone marrow-derived cells (21). However, full activation of naive T cells required the engagement of the TCR in addition to costimulation, the lack of the latter leading to a state of anergy or unresponsiveness. On the other hand, separate delivery of the two signals to normal CD4+ T cells was about 80-fold less efficient than their combined delivery by one cell (9). Ding and Shevach argued that the major factor determining whether cells could deliver trans-costimulation could be the level of B7 expressed on the surface of the APC (10).
We and others previously demonstrated that naive CD8+ T cells could be fully activated by signal-1 alone (15, 22). The requirements for costimulation were shown to depend on both signal-1 concentration and duration. As postulated in the strength of signal-1 model (5), the absence of costimulation can be overcome by high doses of signal-1. Moreover, prolonged signal-1 was shown to generate a functional CD8+ T cell response in vivo in the absence of costimulation (23). In the present work we observed a proliferative response of the cells to short contact time (20 min) with a high concentration of signal-1 in the absence of signal-2, which indicates that at least in vitro, prolonged signal-1 stimulus is not required to activate CD8+ T cells in the absence of costimulation.
The absence of costimulatory signal may also lead to clonal inactivation, either through T cell anergy or cell death by apoptosis (24), and is believed to play a critical role in maintaining self tolerance in vivo (25). When both specific ligand and costimulatory molecules are expressed on the surface of the same cell, naive T cells will clonally expand without threatening tolerance maintenance to tissue-specific self Ag. Conversely, separate encounters with Ag on cells expressing enough signal-2 to induce full T cell activation could become a potential threat to self tolerance. In fact, bystander-presenting (as well as nonpresenting) cells expressing high level of B7 molecules and residing in tissues have been described under particular conditions, such as cytokine mediation (26, 27) and microbial or pathological induction (28, 29). Moreover, small resting B cells that are poor APC for primary responses may become effective APC after the responding T cell population has been activated (29). Such stimulated B cells may, in turn, costimulate more Ag-specific T cells, allowing for an amplification of the specific immune response. If the stimulation originated from infection with a pathogen, an increase in trans-costimulation may lead to a form of autoimmunization. On the other hand, stimulated B cells may costimulate not only Ag-specific T cells, but also non Ag-specific T cells, including self-reactive T cells, thus leading to potential autoimmune responses.
It is difficult to compare our artificial in vitro system based on molecules immobilized on plastic to other in vitro systems where presenting cells, transfected or not, are used. More specifically, instead of CD28 natural ligands CD80 (B7-1) and CD86 (B7-2) as signal-2, we used CD28 mAb, which could trigger a nonphysiological response through the artificial aggregation of the CD28 molecule. However, Levine et al. (30) showed that the kinetics of induction of CD4+ T cell proliferation after anti-CD3 stimulation were similar whether delivered by the natural ligands or by mAb against CD28. Moreover, costimulation delivered by either B7 or anti-CD28 molecules to a T cell hybridoma resulted in the comparable up-regulation of Bcl-xL and prevention of FasL expression (31). Although some signal transduction events may not be shared by B7- and Ab-mediated ligation of CD28, it is possible that full stimulatory potential of the CD28 receptor requires the cross-linking of at least its two natural ligands, while anti-CD28 mAb alone can fully trigger the CD28 receptor (32). We suggest that even though our system is artificial, it may resemble nonordinary or pathological conditions in which CD8+ T cells stimulated by signal-1 encounter only presenting and/or nonpresenting cells that express large amounts of costimulus and independently deliver a signal-2, thus potentiating signal 1.
Ding and Shevach (10) demonstrated that the delayed delivery of B7 costimulation to CD4+ T cells resulted in decreased proliferation. On the other hand, we observed increased proliferation of CD8+ T cells when signal-2 addition was delayed for up to 2 h after signal-1 termination. Thus, we suggest that CD4+ and CD8+ T cells do not respond to delayed costimulation in the same manner. Nevertheless, as has been shown for CD4+ T cells (6), our results indicate that costimulation is not required for the initiation of proliferation of CD8+ T cells.
Costimulation (either concomitant or delayed) of CD8+ T cells activated with signal-1 results in an increase of some precursors, as shown in CFSE-labeled cell experiment. Thus, in our system the proliferation of a whole population upon stimulation after or together with costimulation reflects the acceleration of the response of already recruited T cells due to costimulation. A similar result has been found with total splenocytes (20).
Overall, our data demonstrate that once delivered alone to the CD8+ T cells, signal-1 retains its ability to be potentiated by delayed signal-2. Thus, triggering of the TCR/CD3 complex of CD8+ T cells initiates a cascade of biochemical events that are still sensitive to potent synergy by further signaling. The physiological relevance of this conclusion is interesting: upon specific antigenic stimulation alone, the CD8+ T cell could be considered as in an "excited" state that lasts at least a few hours, during which they may still be sensitive to costimulation. Moreover, reinforcement of signal-1 through the use of a superagonist allows delay between signal-1 and 2 to be extended, which may indicate that the level of excitement of the activated CD8+ T cell depends on the strength of signal-1, but not its duration, and may be modulated. This excited state hypothesis may have important implications not only in terms of immune and autoimmune responses as was discussed above, but also in adoptive immunotherapy, where maintenance of preactivated specific CD8+ T cells could be obtained by providing anti-CD28 mAb separately.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CFSE, carboxy-fluorescein diacetate succinimidyl ester; HA, hemagglutinin.
Received for publication June 14, 1999. Accepted for publication February 16, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), transferred immediately to wells
coated with anti-CD28 mAb at 20 µg/ml (+signal-2, 
), or
transferred to empty wells. After 30 or 120 min, respectively, in the
empty wells, cells were finally transferred to wells coated with
signal-2 (30-min delay, 
, or 120-min delay, 
) and incubated.
After 32 h at 37°C, cells were labeled with
[3H]thymidine for 16 h. Positive control using Con A
at 2.5 µg/ml (conA, 
), as well as negative controls using either
immobilized SC-Kd/CW3 and anti-CD28 mAb (40 and 20 µg/ml,
respectively, irrelevant + signal-2, 
) are presented. Data are the mean
response of triplicate cultures and are representative of three
independent experiments. The signal-1 only experimental values at any
time point are significantly different from the signal-1 plus signal-2
experimental values by paired two-tailed t test, with
p < 0.0001, as well as from the 30 and 120 min
delayed signal-2 experimental values, with p <
0.00001 and p < 0.001, respectively.
). After 32 h at 37°C,
cells were labeled with [3H]thymidine for 16 h. Data
are the mean response of triplicate cultures and are representative of
two independent experiments.
