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* Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands;
Division of Molecular Genetics and Center of Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and
Vaccine Research Center, Emory University, Atlanta, GA 30329
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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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Of the different costimulatory pathways involved in T cell priming, the signal delivered by CD80 and CD86, which are expressed at high levels on mature DC, to the CD28 receptor on T cells has been investigated most extensively (reviewed in Refs. 5 and 6). Although this signal plays a pivotal role in T cell activation, a number of additional costimulatory pathways also contribute to this process. One of these involves the 4-1BB/4-1BB ligand (4-1BBL) receptor-ligand pair. 4-1BB is a member of the TNFR family and is expressed on activated CD8+ and CD4+ T cells (7, 8). Its natural ligand, 4-1BBL, is expressed on B cells, macrophages, and DC (9, 10). In vitro studies have shown that stimulation of T cells with agonistic anti-4-1BB Abs increased TCR-induced proliferation and cytokine production by both CD8+ and CD4+ T cells (7, 11). Accordingly, blocking of 4-1BB costimulation in vitro was shown to inhibit APC-mediated T cell stimulation (12, 13), whereas 4-1BBL-deficient mice showed a reduced capacity to raise CTL immunity against virus infections (14, 15, 16). Furthermore, administration of agonistic anti-4-1BB Ab in mice was shown to amplify the generation of CTL in a murine model for graft vs host disease (11), whereas injection of these Ab into tumor-bearing mice resulted in the rejection of poorly immunogenic tumors (17).
Most published data argue that 4-1BB triggering, rather than being strictly required for induction of T cell immunity, is part of a complex costimulatory signal at the APC/T cell interface involving multiple ligand/receptor pairs. Only the paper by Melero et al. (17) suggests that the presence of a costimulatory signal through 4-1BB may truly make the difference between failure and efficacy of antitumor immunity. This prompted us to investigate the effects of 4-1BB costimulation on the generation of antitumor CTLimmunity in more detail. Our experiments, which study the induction of CTL immunity against Ad5E1 tumor cells, reveal that in vivo triggering of 4-1BB, similar to in vivo triggering of CD40, can indeed make the difference between CTL nonresponsiveness and priming. Both the CD40 signal to APC and the 4-1BB signal to CTL are equally efficient in enabling CTL priming in the absence of CD4+ T cell help. Interestingly, we found that action of this powerful 4-1BB signal requires the CD28-costimulatory pathway to be intact, indicating that 4-1BB signaling is positioned downstream of CD28 costimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All in vitro cultures and assays were performed in IMDM (Life Technologies, Paisley, U.K.) supplemented with 8% FCS, 5 x 10-5 M 2-ME, glutamine, and penicillin (culture medium). C57BL/6 (H-2b) mice were purchased from IFFA Credo (Paris, France).
Vaccinations
Peptides Ad5E1A234–243, Ad5E1B192–200, or the control peptide human papillomavirus 16 E749–57 were dissolved in DMSO and diluted in PBS. For peptide vaccination, mice were injected s.c. with 100 µl of peptide (20 µg) diluted in PBS mixed with 100 µl of IFA. For in vivo triggering through CD40 or 4-1BB, mice received 100 µg of the CD40-activating Ab FGK-45 i.v., or the activating anti-4-1BB Ab 3H3 (11) given i.p. in 200 µl PBS on days 0, 1, and 2 after immunization. In blocking experiments, mice received 200 µg of the control rat IgG2a 6E9 (directed against human gp39; Ref. 18), the anti-4-1BBL Ab 14B3, or CTLA4-Ig i.p. (19) on days 0, 2, 4, 6, and 8 after vaccination. The anti-4-1BBL Ab 14B3 was generated as described (11) by injecting rats with a soluble mouse 4-1BBL-CD8 fusion protein (20).
In cross-priming experiments, mice were injected s.c. with 8–10 x 106 Ad5E1 mouse embryo cells (MEC) derived from TAP knockout or B6 background. These cells are C57BL/6-derived (H-2b) MEC transfected with the Ad5E1 region containing both the E1A and E1B gene (21). Depletion of CD4+ T cells was conducted as follows: mice received 100 µg of the anti-CD4 Ab GK1.5 in 200 µl PBS i.p on days -7, -5, and -3 before Ad5E1 MEC injection and depletion was sustained twice weekly.
Cytotoxicity assay
Splenocytes from immunized mice were cocultivated in 24-well
plates at 5 x 106 cells/well with
IFN-
-treated Ad5E1 MEC at a ratio of 10:1. Following a 6-day
coculture, viable lymphocytes were collected and tested for
cytotoxicity on Europium-labeled target cells as described previously
(21). The mean percentage specific lysis of triplicate
wells was calculated as follows: percentage of specific lysis =
[(cpm experimental release - cpm spontaneous release)/(cpm
maximum release - cpm spontaneous release)] x 100. The
SD of triplicates was in all cases <5%. Target cells used in
cytotoxicity assays are the murine T cell lymphomas RMA and
EL-4, B6 MEC, and Ad5E1 MEC. All target cells are of B6
(H-2b) origin, but only the Ad5E1 MEC express the
Ad5E1A and E1B CTL epitopes.
TCR-transgenic (Tg) T cells
Tg mice on B6 genetic background expressing the TCR derived from a CTL clone that recognizes the Ad5E1A peptide epitope in the context of H-2Db have been described previously (22). Freshly isolated splenocytes from naive TCR-Tg mice (recombination-activating gene+/+ background) were stained with FITC-labeled anti-CD8 Ab and PE-labeled Db/E1A-peptide tetramers (23). The fraction of tetramer-positive T cells averaged between 75 and 95% of the CD8+ population and 12 and 25% of the total splenocyte population. Based on these measurements, a total splenocyte suspension containing 5 x 106 tetramer-positive CD8+ T cells, was injected i.v. per mouse. Where indicated, CFSE-labeling was conducted as follows: splenocytes were isolated and erythrocytes were lysed with NH4Cl. Spleen cells were washed in cold PBS and resuspended at 10 x 106/ml and labeled with 0.5 µM CFSE at 37°C for 30 min. Then FCS was added to a final concentration of 5%. Cells were washed and injected in PBS.
Flow cytometry analysis
For flow cytometry analysis, FITC-, PE-, or APC-conjugated Abs
against CD86, MHC class II, CD11c, CD8
, and IFN- (BD PharMingen,
San Diego, CA), anti-Thy1.2 and goat-anti-rat (Biotrend,
Cologne, Germany), and anti-4-1BBL (14B3) were
used.
For intracellular IFN- detection, spleen cells from immunized mice
were cocultured in vitro with Ad5E1-transformed tumor cells for 6 days.
Viable cells were isolated, and 200,000–500,000 cells per well were
stimulated for 24 h with 1 µg/ml E1B peptide or control peptide
in 96-well plates. During the last 5 h of stimulation, 10 µg/ml
brefeldin A was added. Next, cells were washed in PBA (PBS containing
0.5% BSA, 0.02% azide) and fixed in 4% paraformaldehyde for 10
min (all incubations were done on ice). Cells were washed in PBA
and in PBA containing 0.1% saponin (PBA-sap) and permeabilized in
PBA-sap supplemented with 10% FCS for 10 min. Staining was performed
with Abs diluted in PBA-sap for 30 min, washed in PBA-sap, and fixed in
1% paraformaldehyde. Data acquisition and analysis was performed on a
BD Biosciences FACSCaliber using CellQuest software (BD Biosciences,
Mountain View, CA).
In vitro Ab stimulation
The 96-well plates were coated overnight with various concentrations of anti-CD3 Ab (145.2C11) with or without 5 µg/ml of anti-CD28 Ab (3N7) in PBS. Plates were washed twice with PBS and 200,000–500,000 naive spleen cells or purified CD8+ T cells were added to the wells. After 24 h incubation, the cells were analyzed by FACS.
CD8+ T cells were purified as follows: spleen cells were isolated and B cells were removed with goat anti-mouse IgG magnetic beads according to the manufacturer’s protocol (Polysciences, Warrington, PA). Then cells were incubated with anti-CD4 Ab (GK1.5, 20 µg/ml) and anti-B220 (6B2, 20 µg/ml) for 30 min on ice, washed three times, and incubated with 10x excess of goat-anti-rat IgG magnetic beads (Polysciences) for 30 min on ice and put on a magnet. Remaining nonlabeled cells were >80% CD8+, and were cultured on Ab-coated plates as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that in vivo CD40 triggering of APC
through the administration of an agonistic anti-CD40 Ab can replace
the need for CD4+ Th activity in cross-priming of
Ad5-specific CTL (1). This prompted us to investigate
whether in the same experimental system signals downstream of CD40
triggering that orchestrate the cognate interaction between APC and
CTL, could similarly permit Th-independent cross-priming of CTL. We
injected Ad5E1-transformed MEC into syngeneic
CD4+ T cell-depleted C57BL/6 (B6) mice in
combination with either anti-CD40 or anti-4-1BB Ab. These Ad5E1
MEC were derived from TAP-deficient mice, and therefore, are incapable
of presenting the Ad5E1-encoded CTL epitopes in the context of their
own MHC (21). Consequently, priming of Ad5E1-specific CTL
in this setting relies on cross-presentation of tumor Ags by host APC.
The induction of Ad5E1B-specific CTL is CD4+
Th-dependent, as mice depleted for CD4+ T cells
fail to mount an E1B-specific CTL response (Fig. 1
b). Systemic administration
of agonistic anti-CD40 Ab in these CD4-depleted animals restored
E1B-specific cross-priming (Fig. 1c; Ref. 1).
Interestingly, systemic administration of an agonistic anti-4-1BB
Ab resulted in equally efficient priming of E1B-specific CTL in the
absence of CD4+ T cell help (Fig. 1d).
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, e–g). Similar results were obtained
when E1A-specific CTL immunity was monitored by staining of splenocytes
with H-2Dd/E1A peptide tetramers (not shown).
Thus, in vivo triggering of 4-1BB can prevent the induction of
peptide-specific tolerance, and instead result in the priming of a
potent E1A-specific CTL response. Taken together, these data
demonstrate that the 4-1BB signal efficiently provides CTL with a
license to kill, and as such can replace the need for CD40 triggering
through Th cells or administration of agonistic anti-CD40
Ab. Anti-4-1BB Ab does not induce DC activation
Current knowledge of the expression patterns of CD40 and 4-1BB
would argue that the signal provided by in vivo administration of
anti-CD40 Ab is delivered to the APC, whereas that of
anti-4-1BB Ab is delivered directly to the CTL. To exclude the
possibility that the anti-4-1BB Ab, instead of directly stimulating
Ag-specific T cells, would mediate its effect on CTL priming (Fig. 1)
in an indirect fashion by activation of the Ag-presenting DC, we
investigated whether injection of the anti-4-1BB Ab activates DC in
vivo. B6 mice were injected with anti-4-1BB, anti-CD40, or
control Ab, and the CD86 (B7.2) expression on
CD11c+ DC isolated from the spleens of these mice
was analyzed. As expected (23), in vivo triggering of CD40
resulted in an enhanced expression of CD86, indicating in vivo
activation of the DC. In contrast, after in vivo 4-1BB triggering or
administration of control Ab, the expression of CD86 on DC was not
increased above background (Fig. 2a). The failure of the
anti-4-1BB Ab to induce DC activation is in accordance with the
fact that neither immature nor mature DC express detectable levels of
4-1BB, implicating that DC are not receptive for 4-1BB triggering (Fig. 2b). Therefore, anti-4-1BB Ab does not appear to affect
the DC population, but rather mediates its stimulatory effect on CTL
priming through direct stimulation of CD8+ T
cells.
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To investigate in which manner agonistic anti-4-1BB Ab permits
Th-independent priming of Ad5-specific CTL, we exploited adoptively
transferred T cell populations from Ad5E1A peptide-specific TCR Tg mice
(22). In contrast to the endogenous E1A-specific CTL
response repertoire, the TCR Tg T cells allowed us to determine the
fate of E1A-specific CTL upon immunization with the E1A peptide.
Tracking of these T cells in vivo was performed in two distinct ways.
First, TCR Tg T cells were labeled with CFSE and transferred into
normal B6 mice, after which these mice were vaccinated with the
Ad5E1A-peptide in IFA with or without anti-4-1BB Ab treatment.
Interestingly, the resulting data revealed that proliferation of the
TCR-Tg T cells was equally triggered in mice that had received the E1A
peptide either with or without anti-4-1BB Ab (Fig. 3a). Therefore, anti-4-1BB
Ab do not enable CTL priming by increasing the initial activation or
proliferative capacity of Ag-specific T cells.
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b shows that 11 days
after vaccination, massive expansion of CD8+ T
cells from TCR-Tg origin was observed in mice vaccinated with E1A
peptide in combination with anti-4-1BB Ab, but not in mice that
received either E1A-peptide or anti-4-1BB Ab alone. Therefore,
accumulation of E1A-specific CTL required the presence of the
4-1BB-costimulatory signal. In the absence of this signal, E1A-specific
TCR Tg T cells were ultimately deleted in a manner similar to that
observed for the endogenous E1A-specific CTL repertoire (Fig. 1, e–g, and Refs. 22 and 23).
Furthermore, stimulation of CD8+ CTL immunity
through 4-1BB was dependent on the presence of an Ag-specific trigger.
Analysis of the kinetics of the response of the TCR-Tg T cells reveals
that administration of the antigenic trigger in the presence of 4-1BB
costimulation results in a rapid accumulation of E1A-specific T cells,
which even peaks around day 6 after immunization, followed by a gradual
decline in specific T cell numbers. In contrast, no accumulation of
E1A-specific T cells is detected at any of the time points tested,
indication that T cell deletion in the absence of the 4-1BB signal is a
very rapid and efficient process (Fig. 3c). Taken together,
these data demonstrate that 4-1BB triggering is not essential for
initial CTL activation, but promotes the survival of Ag-stimulated CTL,
thereby permitting expansion of these CTL. Involvement of 4-1BBL/4-1BB interactions in CTL priming
Because in vivo triggering of 4-1BB allows for CTL priming in the
absence of the CD40-dependent DC activation signal (provided by either
Th cells or administration of agonistic anti-CD40 Ab; Fig. 1), this
implies that Ag presentation by nonactivated DC in combination with an
activating anti-4-1BB Ab suffices for efficient CTL priming. The
need for coadministration of anti-4-1BB Ab also indicated that
nonactivated DC were apparently not capable of stimulating CTL through
4-1BB. Therefore, we analyzed expression of the natural ligand for this
receptor, 4-1BBL, on immature and mature DC. Nonactivated DC express
low levels of 4-1BBL, whereas the expression of this molecule is
strongly increased as a result of activation through the CD40 receptor.
This induction of 4-1BBL was observed upon CD40 triggering of DC in
vivo (Fig. 4) and in vitro (data not
shown). This increase in 4-1BBL expression was paralleled by elevated
levels of CD86 on the activated DC. This suggests that both
costimulatory signals contribute to the capacity of mature DC, as
opposed to immature DC, to induce CTL immunity.
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). B6 mice were immunized with
Ad5E1 MEC in combination with systemic doses of anti-4-1BBL Ab
and/or CTLA4-Ig. Interestingly, in vivo blockade of anti-4-1BBL
resulted in a marked reduction in the number of Ag-specific CTL induced
by the tumor cell vaccine. Blockade of CD28 costimulation completely
inhibited CTL induction and, therefore, additional 4-1BBL blocking in
combination with CTLA4-Ig had no extra effect. It should be noted that
the reduction of the amount of CTL induced in the presence of 4-1BBL
blockade was less apparent when analyzed in cytolytic assays, whereas
CD28 blockade resulted in complete absence of target cell lysis. These
data indicate that 4-1BBL/4-1BB interactions play a role in the
cross-priming of Ag-specific CTL, as blocking of the interaction
between these molecules diminishes the numbers of tumor-specific CTL
that are sustained. However, in the absence of the costimulatory signal
through CD28, provided by properly activated DC, the signal provided by
4-1BBL is apparently not capable of enabling CTL priming and, as a
result, induction of antitumor CTL immunity is completely
abrogated.
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The in vivo blocking experiments suggested a dominance of CD28
costimulation over the 4-1BB signal. Therefore, we investigated whether
in the absence of CD28 costimulation, administration of anti-4-1BB
Ab would still provide CTL with a license to kill. B6 mice were
depleted for CD4+ T cells and immunized with
Ad5E1 MEC in combination with administration of anti-4-1BB Ab
and/or CTLA4-Ig. Mice that received anti-4-1BB Ab mounted strong
E1B-specific CTL responses, but mice that also received CTLA4-Ig failed
to do so (Fig. 6). These data indicate
that the positive effect of 4-1BB triggering on the induction of CTL
immunity is dependent on the presence of an intact CD28 costimulatory
signal. Furthermore, these data suggest that the basal levels of
CD80/CD86 expressed by nonactivated DC provide sufficient costimulation
through CD28 to allow additional costimulation through the 4-1BB
pathway. In vitro studies have demonstrated that 4-1BB expression on
naive T cells is absent and that TCR triggering, through cross-linking
by anti-CD3 Ab, results in a rapid increase in 4-1BB surface
expression (7). As our data argue that costimulation
through CD28 is prerequisite for 4-1BB signaling, we investigated
whether CD28 triggering contributes to the up-regulation of 4-1BB on
naive T cells. Therefore, naive total spleen cells or purified
CD8+ T cells were stimulated in vitro with
plate-bound anti-CD3 Ab and analyzed for 4-1BB expression 24 h
later (Fig. 7). It is clear that strong
signals through the TCR (high concentrations of anti-CD3 Ab) are
sufficient to induce 4-1BB expression within 24 h. However, when T
cells are stimulated with lower anti-CD3 concentrations, their
capacity to up-regulate 4-1BB is largely lost (Fig. 7). These lower
anti-CD3 Ab concentrations provide a weaker TCR trigger that is
more likely to resemble an in vivo signal of the kind provided by the
tumor cell vaccine. Importantly, under these weak CD3-triggering
conditions, costimulation through the CD28 receptor restored the
capacity of T cells to express high levels of 4-1BB. These findings are
in accordance with the fact that blockade of the CD28 pathway in vivo
abrogates costimulation through 4-1BB (Fig. 6). Furthermore, they
strengthen the notion that CD28-costimulation of Ag-stimulated T cells
is an important signal for 4-1BB up-regulation on naive T cells,
thereby making these cells susceptible for 4-1BB triggering.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, e–g), and in cross-priming of tumor-specific CTL in the
absence of CD4+ T cell help (Fig. 1, a–d). Furthermore, in vivo blockade of 4-1BBL/4-1BB
interactions markedly decreases the efficacy of Th-dependent
cross-priming of tumor-specific CTL (Fig. 5). Importantly, in vivo
costimulation through CD28 is necessary for effective signaling through
4-1BB to take place (
Figs. 5–7), indicating that the 4-1BB signal lies
downstream of CD28 costimulation.
The effect of the anti-4-1BB Ab on the immunogenicity of the Ad5E1A
peptide vaccine is as striking as the effect we previously reported for
the agonistic anti-CD40 Ab (Fig. 1; Ref. 23). In both
cases, systemic coadministration of the Ab converted the tolerogenic
vaccine into a formulation capable of efficient CTL priming. This
indicates that costimulation through 4-1BB is a promising novel
approach for increasing the potency of vaccines. Importantly, the
effect of in vivo 4-1BB triggering is conceptually distinct from that
of in vivo CD40 triggering. Systemic administration of anti-CD40 Ab
in combination with the E1A peptide vaccine results in the in vivo
activation of the peptide-loaded DC, thereby endowing these DC with the
capacity to prime E1A-specific CTL (23). In a similar
fashion, administration of anti-CD40 Ab was shown replacing the
need for CD4+ T cell help in cross-priming of CTL
by an Ad5 MEC tumor cell vaccine (1). We now show that the
agonistic anti-4-1BB Ab allows for CTL priming in these very same
settings (Fig. 1), but mediates its immunostimulatory action by
directly triggering the CTL, rather than indirectly through activation
of DC (Fig. 2). This is in agreement with previous studies which
revealed that agonistic anti-4-1BB Ab can augment T cell responses
in vitro, in particular of CD8+ T cells (7, 11). Thus, the signal through 4-1BB acts downstream of CD40
signaling, and administration of agonistic anti-4-1BB Ab replaces
the need of CD40-mediated APC activation by either Th cells or by
agonistic anti-CD40 Ab. In accordance with this notion, we found
that activation of DC by inflammatory agents or through CD40 triggering
results in increased 4-1BBL expression (Fig. 4), implicating that only
properly activated DC can provide a strong costimulatory signal to the
4-1BB receptor on T cells.
Analysis of the mechanism by which 4-1BB triggering enables the
induction of CTL immunity revealed that this signal enhances the
survival of Ag-stimulated T cells (Fig. 3). It was previously shown
that triggering through 4-1BB results in the increased survival of
anti-CD3-stimulated T cells in vitro as well as of
superantigen-stimulated T cells in vivo (7, 8). We now
demonstrate that this feature of 4-1BB also applies to the induction of
Ag-specific, MHC-restricted CTL responses in vivo. Notably, the initial
proliferation of T cells upon Ag exposure also occurs in the absence of
4-1BB costimulation (Fig. 3a). This implies that 4-1BB plays
a role more downstream in the process of T cell activation. Indeed, our
experiments, in which we compared the roles of the costimulatory
signals through 4-1BB and CD28, are in support of this. In particular,
agonistic anti-4-1BB Ab failed to permit cross-priming of antitumor
CTL in CD4+ T cell-depleted animals when the
costimulatory signal through CD28 was blocked by CTLA4-Ig
administration (Fig. 6). Furthermore, we show that TCR triggering in
combination with CD28 costimulation is much more efficient in inducing
surface expression of 4-1BB on T cells (Fig. 7) than TCR triggering
alone. Thus, stimulation of naive T cells through their constitutively
expressed TCR and CD28-receptor primes these T cells and at the same
time makes these cells receptive for a survival signal through 4-1BB.
Provision of this signal, either by 4-1BBL expressed on activated DC
(Fig. 4) or by agonistic anti-4-1BB Ab, results in survival, and
thereby, expansion of the Ag-primed T cells (Figs. 1 and 3); whereas
lack of this survival signal will limit the magnitude of these
responses (Fig. 5, Refs. 14, 15, 16).
Whereas our data indicate that delivery of the survival signal through
4-1BB depends on CD28 costimulation (Figs. 6 and 7), others have
reported that 4-1BB triggering of T cells can also take place
independently of the CD28 pathway. However, these studies all examined
the role of the 4-1BB signal in combination with very strong TCR
triggering, such as high concentrations of plate-bound anti-CD3 Ab
or coculture of T cells with allogeneic APC (10, 13, 14, 24). Both our results (Fig. 7) and the data of others
(7) have shown that strong TCR triggering in the absence
of a signal through CD28 suffices to induce 4-1BB expression on naive T
cells, thereby enabling 4-1BB costimulation of T cells independent of
the CD28 pathway. Thus, our data are not in conflict with previously
published reports concerning the relation between the CD28 and 4-1BB
costimulatory pathways in the presence of very strong TCR triggering.
Notably, in our study we have also looked at this relation in settings
where the TCR trigger is weaker. Although high concentrations of
anti-CD3 Ab suffice to induce 4-1BB expression of naive T cells,
lower concentrations of these Ab fail to efficiently up-regulate 4-1BB,
unless costimulation through CD28 is provided (Fig. 7). Accordingly,
Saoulli et al. (25) reported that 4-1BB costimulation of
resting T cells was only found when combined with high amounts of
anti-CD3 Ab, not when combined with lower concentrations of
anti-CD3 Ab. Most importantly, we now demonstrate that the
dependency of the 4-1BB signal on the CD28 pathway also extends to an
in vivo setting where a more physiological signal to the TCR is
provided by a tumor cell vaccine. Although in vivo administration of
agonistic anti-4-1BB Ab permits cross-priming of CTL against this
tumor cell vaccine in the absence of CD4+ T cell
help, this immunostimulatory effect of anti-4-1BB Ab is abrogated
if the CD28 pathway is blocked (Fig. 6). Likewise, the signal provided
by 4-1BBL on properly activated APC in CD4+
Th-proficient mice fails to potentiate CTL priming if CD28
costimulation is blocked (Fig. 5). Therefore, we conclude that under
physiological conditions, the delivery of the 4-1BB-mediated survival
clearly depends on the CD28 pathway.
The fact that blockade of CD28 costimulation completely abolishes CTL
cross-priming (Fig. 6) has interesting implications for our observation
that agonistic anti-4-1BB Ab permits cross-priming in
CD4+ T cell-depleted mice (Fig. 1). These data
argue that even in the absence of CD4+ T cell
help, the cross-presenting APC can provide costimulation through CD28,
which in combination with the antigenic signal is sufficiently strong
to sensitize the CTL for 4-1BB triggering. This is quite conceivable,
as also immature DC express CD80 and CD86, albeit at much lower levels
than their activated counterparts (Fig. 2; Ref. 4).
In summary, stimulation of CTL through 4-1BB is dependent on TCR triggering and CD28 costimulation. Engagement of the 4-1BBL/4-1BB pathway is important for the efficient induction of CTL immunity. Signaling through 4-1BB promotes survival of Ag-triggered CTL, thereby increasing the magnitude of the CTL response after vaccination with either tumor cells or minimal peptide epitopes. The effects of in vivo 4-1BB triggering are particularly dramatic in cases where insufficient CD4+ T cell help is available to properly activate APC. Our observations point at an important role for 4-1BB costimulation in the multistep activation of naive CTL by properly matured DC, and stress the potency of 4-1BB as a license to kill signal for improving the efficacy of vaccines.
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Acknowledgments |
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Footnotes |
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2 Address requests for anti-4-1BB Abs to Dr. Robert Mittler at the current address: Yerkes Primate Center, Department of Microbiology and Immunology, Emory University School of Medicine, 954 Gatewood Road, Atlanta, GA 30329. E-mail address: rmittler{at}rmy.emory.edu
3 Address correspondence and reprint requests to Dr. Rienk Offringa, Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, E3-Q, Albinusdreef 2, 2333 RA Leiden, The Netherlands. E-mail address: R.Offringa{at}lumc.nl
4 Abbreviations used in this paper: Ad5, adenovirus type 5; E1, early region 1; Tg, transgenic; MEC, mouse embryo cell; DC, dendritic cell; 4-1BBL, 4-1BB ligand.
Received for publication August 3, 2001. Accepted for publication January 18, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 M. Stasiolek, A. Bayas, N. Kruse, A. Wieczarkowiecz, K. V. Toyka, R. Gold, and K. Selmaj Impaired maturation and altered regulatory function of plasmacytoid dendritic cells in multiple sclerosis Brain, May 1, 2006; 129(5): 1293 - 1305. [Abstract] [Full Text] [PDF]
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G. Pulle, M. Vidric, and T. H. Watts IL-15-Dependent Induction of 4-1BB Promotes Antigen-Independent CD8 Memory T Cell Survival. J. Immunol., March 1, 2006; 176(5): 2739 - 2748. [Abstract] [Full Text] [PDF]
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V. Y. Taraban, T. F. Rowley, and A. Al-Shamkhani Cutting Edge: A Critical Role for CD70 in CD8 T Cell Priming by CD40-Licensed APCs J. Immunol., December 1, 2004; 173(11): 6542 - 6546. [Abstract] [Full Text] [PDF]
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P. Bansal-Pakala, B. S. Halteman, M. H.-Y. Cheng, and M. Croft Costimulation of CD8 T Cell Responses by OX40 J. Immunol., April 15, 2004; 172(8): 4821 - 4825. [Abstract] [Full Text] [PDF]
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R. A. Wilcox, K. Tamada, D. B. Flies, G. Zhu, A. I. Chapoval, B. R. Blazar, W. M. Kast, and L. Chen Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo Blood, January 1, 2004; 103(1): 177 - 184. [Abstract] [Full Text] [PDF]
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C. Wiethe, K. Dittmar, T. Doan, W. Lindenmaier, and R. Tindle Provision of 4-1BB Ligand Enhances Effector and Memory CTL Responses Generated by Immunization with Dendritic Cells Expressing a Human Tumor-Associated Antigen J. Immunol., March 15, 2003; 170(6): 2912 - 2922. [Abstract] [Full Text] [PDF]
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 V. Douin-Echinard, J.-M. Peron, V. Lauwers-Cances, G. Favre, and B. Couderc Involvement of CD70 and CD80 intracytoplasmic domains in the co-stimulatory signal required to provide an antitumor immune response Int. Immunol., March 1, 2003; 15(3): 359 - 372. [Abstract] [Full Text] [PDF]
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J. W. Lillard Jr, U. P. Singh, P. N. Boyaka, S. Singh, D. D. Taub, and J. R. McGhee MIP-1alpha and MIP-1beta differentially mediate mucosal and systemic adaptive immunity Blood, February 1, 2003; 101(3): 807 - 814. [Abstract] [Full Text] [PDF]
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 Z.-Q. Wu, A. Q. Khan, Y. Shen, K. M. Wolcott, W. Dawicki, T. H. Watts, R. S. Mittler, and C. M. Snapper 4-1BB (CD137) Differentially Regulates Murine In Vivo Protein- and Polysaccharide-Specific Immunoglobulin Isotype Responses to Streptococcus pneumoniae Infect. Immun., January 1, 2003; 71(1): 196 - 204. [Abstract] [Full Text] [PDF]
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P. Bansal-Pakala and M. Croft Defective T Cell Priming Associated with Aging Can Be Rescued by Signaling Through 4-1BB (CD137) J. Immunol., November 1, 2002; 169(9): 5005 - 5009. [Abstract] [Full Text] [PDF]
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D. Laderach, M. Movassagh, A. Johnson, R. S. Mittler, and A. Galy 4-1BB co-stimulation enhances human CD8+ T cell priming by augmenting the proliferation and survival of effector CD8+ T cells Int. Immunol., October 1, 2002; 14(10): 1155 - 1167. [Abstract] [Full Text] [PDF]
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), the human papillomavirus 16 E7 peptide (
), or Ad5E1 MEC (
).
The stimulatory effects of 4-1BB Ab on Ad5-specific CTL priming by
tumor cells or the E1A peptide have each been observed in three
separate experiments. Overall, we found that anti-CD40 and
anti-4-1BB Ab were equally capable of enabling Ad5-specific CTL
priming under these conditions.
