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Departments of
*
Pathology and
Neurology, and
Committee in Immunology, University of Chicago, Chicago, IL 60637; and
Department of Immunology, Mayo Clinic, Rochester, MN 55905
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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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In vitro studies have shown that 4-1BB signaling augments T cell
proliferation and cytokine production through both CD28-dependent and
-independent mechanisms (4, 5, 7, 12, 13, 14). Furthermore,
agonistic anti-4-1BB mAbs preferentially stimulate proliferation of
and IFN-
production by CD8+ T cells as
compared with CD4+ T cells (15),
suggesting that 4-1BB is a costimulatory molecule primarily for
CD8+ T cells. In vivo experiments using agonistic
4-1BB mAbs or 4-1BBL-transfected tumor cells have shown that signaling
through 4-1BB can induce the preferential expansion of
CD8+ CTLs that recognize and reject tumors and
allograft transplants (15, 16, 17). Agonistic anti-4-1BB
mAb also prevents superantigen-induced T cell death, especially of
CD8+ T cells (18). In addition, the
study of immune responses in 4-1BBL-deficient mice showed that
CD8+ T cell responses to virus infections were
reduced (19, 20). It appears that activation via 4-1BB is
required for optimal CD8+ T cell-mediated immune
responses in vivo.
The function of 4-1BB in CD4+ T cell-mediated immunity has been explored, with most studies suggesting that it plays a costimulatory role (10, 12, 21, 22, 23). A previous study showed that 4-1BBL could provide a costimulatory signal for T cell activation on APCs lacking B7 molecules by using a MHC class II-restricted autoreactive T cell hybridoma and purified CD4+ T cells (10). Another study used purified CD4+ T cells responding to allogeneic 4-1BBL-expressing stimulator cells to demonstrate that 4-1BB ligation could augment CD28-independent cytokine production by T cells (12). Pigeon cytochrome c-presenting fibroblast APCs that were transfected with 4-1BBL were used to suggest that 4-1BB engagement on naive CD4+ T cells promotes proliferation, cell cycle progression, and IL-2 secretion (21). In addition, both CD4+ and CD8+ T cell-mediated in vivo alloresponses were regulated by 4-1BB/4-1BBL interactions to approximately the same extent (22). 4-1BBL induced cell division and enhanced CD4+ and CD8+ T cell effector functions with similar efficacy when using purified TCR-transgenic T cells responding to a specific peptide and allogeneic stimulator cells that express 4-1BBL (23).
However, the introduction of the same agonistic anti-4-1BB mAb (1D8 and 3E1) clones that were used to promote tumor rejection and enhancing graft-vs-host disease (GVHD) abrogated T cell-dependent humoral immune responses in vivo, possibly by inducing Th cell anergy (24). To further investigate the role of 4-1BB signaling in CD4+ T cell-mediated immune responses and its effects on autoimmune diseases, we studied its function in the development of experimental autoimmune encephalomyelitis (EAE). EAE is a Th type 1 cell-mediated demyelinating disease of the CNS that is often used as an animal model for human multiple sclerosis. It can be induced in several strains of animals by immunization with various myelin proteins or immunodominant peptide epitopes derived from myelin basic protein, proteolipoprotein (PLP), or myelin oligodendrocyte glycoprotein (MOG) (25, 26). Th1-type responses are believed to be responsible for EAE pathogenesis, whereas Th2 responses appear to be protective (27, 28). Because the immunodominant autoreactive Ag in this disease has been identified, EAE serves as an excellent model for studying the function of costimulatory molecules in an Ag-specific system.
Interestingly, we found that the administration of an agonistic anti-4-1BB mAb reduced the incidence and severity of EAE by preventing autoreactive T cell immune responses. Taking advantage of DO11.10 TCR-transgenic mice, we were able to monitor Ag-specific CD4+ T cells, and we found that the administration of anti-4-1BB mAb initially promotes CD4+ T cell proliferation but subsequently accelerates their activation-induced cell death (AICD). Therefore, the engagement of 4-1BB by an agonistic Ab may provide a novel approach to effectively depleting autoreactive T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 10-wk-old C57BL/6, SJL, and BALB/c female mice were purchased from the National Cancer Institute (Frederick, MD). C57BL/6 RAG-1-/- male mice were purchased from The Jackson Laboratory (Bar Harbor, ME). DO11.10 BALB/c mice were kindly provided by Dr. A. Sperling (University of Chicago, Chicago, IL). All mice were housed in the University of Chicago Animal Care Facilities. In each experiment, age-matched mice were used.
Peptides
MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) and PLP139–151 (HSLGKWLGHPDKF) were synthesized by Alpha Diagnostic International (San Antonio, TX). The peptides were >76% pure as determined by HPLC. Chicken OVA323–339 (ISQAVHAAHAEINEAGR) peptide was synthesized by the Divisional Protein-Peptide Core Facility of the University of Chicago. The peptides were >86% pure as determined by HPLC.
Antibodies
Agonistic anti-4-1BB Ab (2A and IgG2a) was generated by immunizing a Lewis rat (Harlan Sprague-Dawley, Indianapolis, IN) with mouse 4-1BBIg.4 Hybridomas were produced by fusing rat spleen cells with mouse Sp2/0 myeloma cells. The culture supernatants were screened by ELISA, and the hybridoma secreting the mAb 2A was selected for additional experiments. To prepare 4-1BBIg fusion protein, the extracellular domain of mouse 4-1BB was amplified from activated spleen cell cDNA using sequence-specific primers and was then fused to the CH2-CH3 domain of mouse IgG2a in an expression plasmid pmIgV, which was transfected into Chinese hamster ovary cells. The protein in the culture supernatants was purified using a HiTrap Protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) and was dialyzed in LPS-free PBS.
mAb 2A stained >80% of purified T cells that had been activated for 24 h by anti-CD3 and anti-CD28 mAbs. Binding is specific, as Ab binding could be competitively inhibited by inclusion of 4-1BB-Ig, whereas inclusion of a control rat IgG Ab did not inhibit binding. Furthermore, mAb 2A binds specifically to the mouse T cell lymphoma S49.1 that constitutively expresses 4-1BB, as demonstrated by another anti-4-1BB mAb, clone 1AH2. Similar to previously reported agonistic anti-4-1BB mAb (1D8 and 3E1; Ref. 15), mAb 2A enhanced T cell proliferation in a dose-dependent fashion in the presence of a suboptimal dose of anti-CD3 mAb in vitro, and it induced rejection of well-established EL4E7 tumor in vivo.4
Rat IgG was purchased from Sigma-Aldrich (St. Louis, MO) and served as a control Ab. The KJ1-26 mAb, which binds only to the particular transgenic TCR heterodimer in DO11.10 TCR-transgenic mice, was kindly provided by Dr. A. Sperling.
Induction of EAE and mAb treatment
To induce EAE by active immunization, female C57BL/6 mice were immunized s.c. at four sites on the flank, with a total of 100 µg of MOG35–55 peptide emulsified in an equal volume of CFA (Life Technologies, Gaithersburg, MD) containing 1 mg/ml Mycobacterium tuberculosis H37 RA (Difco, Detroit, MI) on days 0 and 7. Mice also received i.v. injections of 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2 after immunization. Mice were administered with anti-4-1BB or control rat IgG (or PBS) i.p. on day 0. Because the doses of anti-4-1BB from 100 to 300 µg/mouse showed similar effects, in most experiments, the dose of anti-4-1BB and control rat IgG was used at 150 µg/mouse. For adoptively transferred EAE, draining lymph node (DLN) cells were harvested from female C57BL/6 mice on day 10 post-s.c. immunization with 100 µg of MOG35–55 emulsified in CFA. The cells (5 x 106 cells/ml) were cultured with MOG35–55 peptide (25 µg/ml) for 4 days and were then i.v. transferred into sublethally (600 rad) irradiated female C57BL/6 mice (2 x 107 cells per mouse). The recipients received 200 ng of pertussis toxin by i.v. injection immediately after cell transfer and 2 days later. Recipients were also treated i.p. with anti-4-1BB or control rat IgG on the day of transfer. In some experiments, C57BL/6 mice were immunized s.c. with 100 µg of MOG35–55 emulsified in CFA twice at a 1-wk interval and treated with anti-4-1BB or control IgG on the day of the first immunization. One week after the second immunization, DLN cells and splenocytes were isolated, pooled, and directly transferred into male RAG-1-/- mice.
For induction of the relapsing-remitting EAE model, female SJL mice were immunized s.c. once with 100 µg of PLP139–151 emulsified in CFA containing 500 µg of M. tuberculosis H37 RA at three sites on the flank. On the same day or after the onset of disease, mice were administered 200 µg of anti-4-1BB or control rat IgG i.p once or once a week for a total of 3 wk. Mice were scored daily for signs of EAE according to the following clinical scoring system: 0, no clinical signs; 0.5, partial loss of tail tonicity; 1, complete loss of tail tonicity; 2, flaccid tail and abnormal gait; 3, hind leg paralysis; 4, hind leg paralysis with hind body paresis; 5, hind and fore leg paralysis; and 6, death.
DTH assay
Female C57BL/6 mice were immunized s.c. at four sites on the flank, with a total of 100 µg of MOG35–55 peptide emulsified in an equal volume of CFA on days 0 and 7. Mice also received an i.v. injection of 200 ng of pertussis toxin on days 0 and 2 after immunization. Mice were administered 150 µg of anti-4-1BB or control rat IgG i.p. on day 0. Six weeks after the first immunization, mice were injected in the footpad with 15 µg of MOG in 15 µl of PBS. The thickness of the footpad was measured 24 h after injection. Naive mice were used as negative controls.
Preparation of tissue for histologic examination
Mice were anesthetized with pentobarbital and were perfused by intracardiac puncture with 50 ml of Trump’s fixative (phosphate-buffered 4% paraformaldehyde and 1% glutaraldehyde, pH 7.2). Spinal cords were removed, paraffin-embedded, sectioned coronally, and stained with H&E for visualization of inflammatory infiltrates (29).
In vitro proliferation assay
Ten days after immunization, DLN cells were isolated and cultured in 96-well flat-bottom plates at a concentration of 5 x 105 cells/well in complete RPMI 1640 medium (Life Technologies) that contained 10% heat-inactivated FCS, 1 mM glutamine, 1% penicillin-streptavidin, 1 mM nonessential amino acids, and 5 x 10-5 M 2-ME with various concentrations of MOG35–55 peptide. Plates were pulsed with [3H]thymidine (Amersham Pharmacia Biotech) at 0.5 µCi/well on day 4 of culture for the final 18 h. Incorporation of thymidine into DNA was measured by liquid scintillation counting, and the mean was calculated from triplicate wells.
Detection of cytokine production by ELISA
Four and 10 days after immunization, DLN cells were isolated and
cultured with or without MOG35–55 peptide (25
µg/ml) in complete RPMI 1640 medium for 2 or 3 days. Supernatants
were collected for the detection of IFN-, GM-CSF, IL-10, and IL-4 by
ELISA according to the manufacturer’s instructions. All anti-mouse
cytokine mAbs and recombinant cytokine standards were purchased from BD
PharMingen (San Diego, CA). All cytokine levels were calculated using
standard curves with known amounts of recombinant cytokines.
Real-time PCR
Ten days after immunization, DLNs were removed and total RNA was isolated using TRI reagent (Life Technologies) and was then treated with DNase I (Amersham Pharmacia Biotech). Total RNA (5 µg) was reverse transcribed using the First Strand cDNA Synthesis kit (Amersham Pharmacia Biotech). The mRNA of cytokines and GAPDH were detected by real-time RT-PCR using the ABI Prism 7700-sequence detection system (Applied Biosystems, Foster City, CA) as described previously (30). The primer and probe sequences have previously been reported (31). The probes for cytokines or GAPDH were labeled with 6-carboxy-fluorescein or tetrachloro-6-carboxy-fluorescein, respectively, as a reporter dye. All reactions were conducted in triplicate 20-µl reaction volumes containing TaqMan Universal PCR master mixture (Applied Biosystems). PCR amplification conditions were as follows: 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. PCR results were analyzed using the relative standard curve method according to the manufacturer’s instructions. The amount of cytokine mRNA in each sample was corrected for GAPDH. Final results are displayed as the ratio of the amount of cytokine mRNA over that of control group.
5,6-Carboxy-succinimidyl-fluoresceine-ester labeling of DO11.10 cells
Before fluorescence labeling, TCR-transgenic lymph node and spleen cells from DO11.10 transgenic mice were isolated, and CD3+ T cells were purified by immunomagnetic column by negative depletion method (StemCell Technologies, Vancouver, British Columbia, Canada). The purified T cells containing >90% of CD4+KJ126+ cells were resuspended in PBS at 2 x 107 cells/ml and they were incubated with 10 µM 5,6-carboxy-succinimidyl-fluoresceine-ester (Molecular Probes, Eugene, OR) at 37°C for 30 min.
Tracing adoptively transferred cells
To trace Ag-specific T cells in vivo, we took advantage of DO11.10 TCR-transgenic mice (32, 33). CFSE-labeled and unlabeled TCR-transgenic lymph node and spleen cells from DO11.10 transgenic mice were isolated, and the percentage of CD4+KJ1-26+ cells was measured by flow cytometric analysis. Three to five million CD4+KJ1-26+ cells were i.v. injected into nonirradiated BALB/c mice. On the same day, recipient mice were s.c. injected with OVA323–339 (300 µg) emulsified in CFA in a volume of 0.1 ml distributed among three sites on the hind back and treated with anti-4-1BB or control IgG (150 µg/mouse) i.p. DLN cells from BALB/c recipients of CFSE-labeled DO11.10 cells were isolated 54 h after immunization and analyzed by flow cytometry. DLN cells from BALB/c recipients of nonlabeled cells were isolated 4, 9, and 19 days after immunization and incubated with anti-FcR mAb (2.4G2; American Type Culture Collection, Manassas, VA) in staining buffer (PBS containing 1% FCS and 0.1% sodium azide) on ice for 10 min to block FcR. FITC-labeled anti-CD4 (BD PharMingen) and biotinylated KJ1-26 mAb were then incubated with the cells at 4°C for 20 min. After a wash with staining buffer, the cells were incubated with PE-labeled streptavidin (Immunotech, Luminy, France) at 4°C for 20 min. Following one wash, cells were collected on a FACScan (BD Biosciences, Mountain View, CA) and were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA).
Detection of apoptosis
Four to 5 days after immunization, DLN cells from immunized BALB/c recipients were isolated and stained with biotin-labeled KJ1-26 mAb followed by PE-labeled streptavidin. Following two washes, the cells were stained with either FITC-labeled annexin V (BD PharMingen) or 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) and then analyzed by flow cytometry. For staining with 7-AAD, cells were incubated with FACS buffer containing 20 µg/ml 7-AAD at 4°C for 20 min. The fluorescence of 7-AAD was detected by red channel FL-3 of FACScan. KJ1-26-positive cells were gated to analyze annexin V and 7-AAD staining.
Statistical analysis
Comparison of the mean peak disease severity between two groups of mice was analyzed by the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address the role of 4-1BB in the development of EAE, C57BL/6
mice were treated with either agonistic anti-4-1BB mAb (2A) or rat
IgG control i.p. on the day of the first s.c. immunization with
MOG35–55 peptide emulsified in CFA.
Interestingly, the results showed that 2A treatment greatly inhibited
the development of EAE and reduced the disease incidence and severity
(Fig. 1
A; Table I). All of the control mice developed
moderate (2 of 12; clinical score, 0.5–2) to severe (10 of 12; 83.3%;
clinical score, 3–4) EAE. Recovery from the disease was not observed
in any of the control mice up to 30 days after onset, and similar
results were obtained when the mice were treated with PBS (data not
shown). In contrast, in the 2A-treated group, 5 of 12 mice (41.7%)
were entirely asymptomatic, whereas the rest (7 of 12; 58.3%) only
displayed moderate symptoms (clinical score, 0.5–2). More importantly,
complete recovery was observed in virtually all the mildly symptomatic
mice 10–15 days after disease onset. The mean maximal clinical score
of 2A-treated mice was significantly lower than that of the control
group (0.4 for the 2A-treated group vs 3.1 for the control group;
p < 0.001).
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B). In
contrast, no or only minimal perivascular lymphocytic infiltrate was
observed in the sections obtained from mice treated with 2A (Fig. 1B). These results suggest that 4-1BB engagement during the
priming stage of an immune response strongly inhibits the infiltration
of autoreactive T cells in the CNS and EAE development. Anti-4-1BB mAb treatment does not prevent adoptively transferred EAE
The mechanism by which anti-4-1BB mAb administration inhibits
EAE development may be due to the inhibition of encephalitogenic T cell
responses and/or their migration into the CNS. To dissect this issue,
we tested the effect of 2A treatment in an adoptive transfer model of
EAE. Activated MOG-specific T cells were obtained by coculture for 4
days of MOG35–55 peptide with DLN cells from
C57BL/6 mice immunized with MOG35–55 peptide
emulsified in CFA. EAE was established by transferring these activated
MOG-specific T cells by i.v. injection into sublethally irradiated
C57BL/6 mice that were then treated i.p. with either 2A or control rat
IgG at 200 or 500 µg/mouse. The results showed that the
administration of anti-4-1BB mAb at the time of adoptive transfer
did not prevent EAE induced by activated effector autoreactive T cells
(Fig. 2). This suggests that
anti-4-1BB mAb treatment upon adoptive transfer does not prevent
the trafficking of activated autoreactive T cells into the CNS, and
that 4-1BB plays a role before the effector phase of EAE
pathogenesis.
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To confirm that anti-4-1BB mAb administration inhibits
encephalitogenic T cell responses, pooled DLN cells and splenocytes
from C57BL/6 mice, which were immunized s.c. with
MOG35–55 peptide in CFA and treated i.p. with
control rat IgG or 2A, were adoptively transferred by i.v. injection
into syngeneic male RAG-1-/- mice. The
recipients of control IgG-treated lymphocytes developed severe EAE, and
in a representative experiment, all control recipients died from EAE
9–12 days after adoptive transfer. In contrast, none of the mice that
received 2A treatment developed clinical symptoms by the time the
experiment was terminated (2 mo after transfer; Fig. 3A and data not shown). These
results suggest that the administration of agonistic anti-4-1BB mAb
inhibits autoreactive T cell responses.
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. Mice were then restimulated in the footpad
with MOG35–55 peptide 6 wk after the first
immunization, and 24 h later footpad thickness was measured. Naive
mice were used as negative controls. As shown in Fig. 3B,
control IgG-treated mice, in contrast to naive and 2A-treated mice,
responded to the MOG peptide. Therefore, 2A treatment inhibited
delayed-type hypersensitivity (DTH) responses to MOG peptide,
suggesting that anti-4-1BB mAb treatment inhibits the function of
Ag-specific T cells.
To further address this possibility, we tested the ability of DLN cells
to proliferate in response to MOG peptide stimulation in vitro. DLN
cells were isolated from mice 10 days after s.c. immunization with
MOG35–55 peptide and i.p. injection of either
control rat IgG or 2A. The cells were restimulated with various doses
of MOG35–55 peptide in vitro. As shown in Fig. 4A, DLN cells from control rat
IgG-treated mice proliferated in a dose-dependent manner to
MOG35–55 peptide stimulation, whereas DLN cells
from 2A-treated mice responded poorly. This result further supports our
in vivo results that anti-4-1BB mAb treatment inhibits the function
of autoreactive MOG-specific T cells.
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is an
important Th1 cytokine, and the administration of agonistic
anti-4-1BB Ab was reported to enhance the production of IFN-.
Therefore, we determined the potential effects of in vivo
anti-4-1BB mAb treatment on MOG-specific production of IFN-.
C57BL/6 mice were immunized s.c. with MOG35–55
peptide emulsified in CFA and were treated i.p. with 2A or rat IgG. Ten
days later, DLN cells were isolated and cultured in medium alone or
medium supplemented with MOG35–55 peptide (25
µg/ml) for 2 days. The supernatants were collected, and IFN-
production was determined by ELISA. When cultured with
MOG35–55 peptide, DLN cells from mice treated
with control rat IgG produced high levels of IFN- (Fig. 4B). In contrast, there was a significant decrease in
IFN- production by DLN cells from mice treated with 2A. The levels
of IFN- production by DLN cells cultured with medium alone were very
low (data not shown). To determine whether this differential response
was due to in vitro artifacts, total RNA was extracted from DLN cells
isolated 10 days after immunization for quantification of IFN- mRNA
using real-time quantitative RT-PCR. As shown in Fig. 4C,
expression of IFN- mRNA by DLN cells was significantly reduced in
2A-treated mice. These data demonstrate that 4-1BB engagement results
in the reduced production of the Th1 cytokine, IFN-. To study
whether 2A treatment results in increased production of Th2 cytokines,
IL-4 and IL-10 production by DLN cells was also measured by ELISA. We
found that similar low levels of IL-4 was produced by DLN cells from
mice treated with either control rat IgG or 2A, and IL-10 production
was low and variable within the two groups (data not shown). These
results suggest that agonistic anti-4-1BB does not preferentially
induce Th2 cytokines, but instead inhibits a Th1 response to protect
the mice from the development of EAE pathogenesis. Anti-4-1BB mAb treatment does not inhibit the initial expansion of CD4+ T cells, but promotes AICD
The inhibition of Ag-specific CD4+ T cell
responses by anti-4-1BB treatment could be due to induction of
either anergy or deletion. To discriminate between these two
possibilities, we monitored in vivo Ag-specific responses using
TCR-transgenic T cells. T cells from DO11.10 TCR-transgenic mice
(32), which express transgenes encoding a TCR specific for
chicken OVA peptide 329–339 bound to I-Ad class
II MHC molecule (34), were transferred i.v. into BALB/c
mice and were detected with mAbs specific for CD4 and the clonotypic
TCR (KJ1-26; Ref. 35). To determine whether the
anti-4-1BB mAb (2A) we used in this study inhibited
CD4+ T cell immune responses by preventing the
activation of CD4+ T cells, we transferred
partially purified CFSE-labeled DO11.10 TCR-transgenic T cells into
wild-type BALB/c recipients. The recipients were immunized s.c. with
OVA323–339 peptide emulsified in CFA and were
injected i.p. with 2A or control rat IgG. Fifty-four hours later, the
fluorescence of transgenic cells in DLN was analyzed by flow cytometry.
The results showed that in 2A-treated mice, >70% of the transgenic
cells divided more than five generations compared with 45% doing so in
control mice (Fig. 5A). These
results suggest that anti-4-1BB mAb treatment does not inhibit, but
actually promotes, the proliferation of CD4+
transgenic T cells shortly after immunization.
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, DLNs from both control and 2A-treated mice
contained much higher percentages (Fig. 5B) and numbers
(Fig. 5C) of
CD4+KJ1-26+ cells 4 days
postimmunization with OVA peptide compared with those from unimmunized
recipients. This also suggests that anti-4-1BB mAb administration
does not inhibit the initial proliferation of transgenic T cells in
response to OVA323–339 peptide immunization in
vivo. However, the percentages and numbers of
CD4+KJ1-26+ cells in the
DLNs of 2A-treated mice decreased more rapidly on days 9 and 19 than in
control mice (Fig. 5, B and C). Furthermore,
analysis of CD4 and KJ1-26 double-positive cells in the spleen and
mesenteric lymph nodes revealed similar results (data not shown). These
results indicate that although anti-4-1BB mAb administration does
not inhibit the initial proliferation of transgenic T cells in response
to OVA323–339 peptide stimulation, it does
facilitate the clearance of activated T cells, and therefore does not
appear to induce anergy.
To dissect how anti-4-1BB mAb treatment increases the clearance of
activated T cells, BALB/c recipients of DO11.10 T cells were immunized
and treated as above. Four to 5 days later, DLN cells were isolated and
stained for KJ1-26 and annexin V or 7-AAD, markers for the early stage
of apoptosis and live/dead discrimination, respectively. The results
showed a significant increase in the percentages of apoptotic cells in
2A-treated mice compared with controls (16% vs 6% by staining with
annexin V and 19% vs 7% by staining with 7-AAD (Fig. 5D)).
Therefore, anti-4-1BB mAb treatment increases AICD of Ag-specific
CD4+ T cells, which could serve as a potential
mechanism that regulates the effector Ag-specific T cells.
Anti-4-1BB mAb treatment inhibited MOG-specific Th1 cytokine production at 10 days, but not 4 days, postimmunization
Our study in CD4+ TCR-transgenic T cells
showed that agonistic anti-4-1BB mAb treatment initially promotes
the proliferation and subsequently increases the depletion of activated
CD4+ T cells by enhancing AICD. To determine
whether this also occurs in an EAE model, we examined the function of
MOG peptide-specific CD4+ T cells, 4 and 10 days
postimmunization and Ab treatment, by detecting Th1 cytokine production
in DLN cells. The results showed that 4 days postimmunization,
2A-treated DLN cells produced higher levels of IFN- and GM-CSF than
control DLN cells when cultured with MOG peptide in vitro for 3 days
(Fig. 6, A and C).
However, 10 days postimmunization, 2A-treated DLN cells produced much
less IFN- and GM-CSF than control DLN cells (Fig. 6, B
and D). This demonstrates that agonistic anti-4-1BB mAb
treatment initially promotes the activation of MOG-specific T cells and
then down-regulates their effector functions, most likely by enhancing
AICD of autoreactive CD4+ T cells.
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The relapsing-remitting clinical course is a characteristic
feature of multiple sclerosis. Immunization of SJL mice with
PLP139–151 peptide in CFA results in the
development of chronic relapsing-remitting EAE. To test whether the
administration of anti-4-1BB mAb alters the development of the
relapsing disease, SJL mice were treated with 2A or control rat IgG
once on the day of immunization or once a week for a total of three
times after disease onset. As expected, mice from the control group
developed the typical relapsing-remitting EAE. However, 2A treatment on
the day of immunization significantly reduced the severity of disease
(Fig. 7A). When treatment was
initiated after disease onset, EAE relapse was inhibited (Fig. 7B). These results further confirmed that anti-4-1BB mAb
administration induces the amelioration of EAE in various mouse models,
and, more importantly, can prevent disease relapse.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-4-1BB mAb administration may promote T cell clearance by complement- or FcR-mediated depletion of the activated T cells that express 4-1BB, by blockade of 4-1BB-4-1BBL interactions, and by deliverance of an agonistic signal through the 4-1BB receptor. A high dose of anti-4-1BB Ab (2A) Fab (500 µg/injection) enhanced tumor immunity, suggesting that the Fc fragment that initiates complement activation or that FcR-mediated depletion is not essential for T cell costimulation (L. Chen, unpublished data). In addition, we observed that the same anti-4-1BB Ab prolonged CD8+ T cell activation while increasing CD4+ T cell apoptosis, which is inconsistent with a nonspecific cytolysis mechanism. Furthermore, we could detect 4-1BB-expressing cells 4 days postimmunization in the DLNs of mice injected with MOG peptide and treated with 2A (data not shown). Therefore, we believe that 2A does not cause complement- or Fc-mediated depletion of T cells expressing 4-1BB.
2A could also inhibit CD4+ T cell responses by blocking the signals between 4-1BBL and 4-1BB expressed on activated T cells. We found that EAE mice treated with anti-4-1BBL mAb could not inhibit MOG-specific T cell responses (data not shown). This strongly suggests that just blocking 4-1BB-4-1BBL interactions may not be sufficient to prevent the activation of MOG-specific T cells, and, therefore, 2A must be functioning through some other mechanism.
Our data support the third possibility that 2A delivers an agonistic
signal through 4-1BB, as demonstrated by increased in vivo
proliferation of DO11.10 TCR-transgenic T cells (Fig. 5A)
and augmented in vivo cytokine production by MOG-specific T cells
shortly after immunization (Fig. 6, A and C).
Although the agonistic signal through 4-1BB was able to initially
activate CD4+ T cells, we unexpectedly observed
that it subsequently increased their clearance (Fig. 5, B
and C). Therefore, we have demonstrated that 4-1BB can
costimulate CD4+ T cells and then promote their
AICD. Our results differ from previously published data that
demonstrated that 4-1BB signaling provides a survival signal for
activated lymphocytes (18, 23, 36). One possible
explanation is that various T cell subsets could differentially respond
to 4-1BB-mediated costimulation in vivo. It has been shown that the
administration of agonistic anti-4-1BB mAb induces CD8-mediated
rejection of established tumors and enhances allograft rejection and
GVHD (15, 16). It appears that anti-4-1BB-mediated
CD8+ T cell responses last longer than
CD4+ T cells do (15). Takahashi et
al. (18) demonstrated that 4-1BB ligation is much more
effective in preventing the death of superantigen-activated
CD8+ T cells than CD4+ T
cells in vivo. Our study clearly shows that treatment with agonistic
anti-4-1BB mAb promotes the deletion of activated
CD4+ T cells. Taken together, these results
suggest that 4-1BB engagement may preferentially provide survival
signals for CD8+ T cells in vivo.
In accordance with the majority of studies, we found that 4-1BB ligation by an agonistic mAb promotes the activation of CD4+ T cells by increasing their ability to proliferate and produce cytokines. By using the TCR-transgenic DO11.10 mice, Cannon and colleagues (23) demonstrate that 4-1BBL promotes CD4+ T cell survival in vitro. However, in this study we used DO11.10 mice and observed the dual roles of 4-1BB on CD4+ T cell-mediated immune responses in vivo. 4-1BB initially provided T cells with costimulatory signals, and then it unexpectedly decreased their longevity via AICD. This discrepancy can be attributed to the complex nature of an in vivo immune response.
Our result is in contrast to the studies by Blazar and colleagues (22), which showed that CD4+ T cell-mediated alloresponses were enhanced by agonistic anti-4-1BB mAb. This could be due to differences among various immune responses. During an allogenic T cell response in which MHC molecules are mismatched across the MHC, a high proportion of T cell repertoires are involved, and the response could be more acute than the Ag-specific autoimmune disease model we used. Therefore, 4-1BB may have several functions that depend on the activation status of the cell, subset of cell involved, and the type of immune response initiated. Further studies are needed to elucidate mechanisms by which anti-4-1BB stimulation promotes the AICD of CD4+ T cells in vivo.
In summary, we were able to inhibit autoreactive T cell responses, and therefore prevent EAE pathogenesis, by treating mice with an agonistic anti-4-1BB mAb. The down-regulation of autoreactive T cell responses could be due to regulation of the Th1/Th2 balance, activation of various subsets of regulatory cells, induction of T cell unresponsiveness (anergy), or deletion of activated cells via AICD. In this study, we have demonstrated that 4-1BB does not appear to affect the Th1/Th2 balance or anergize CD4+ T cells. Instead, we have clearly shown that the administration of agonistic anti-4-1BB mAb initially increases T cell activation and then promotes the clearance of these activated CD4+ T cells, which results in the attenuation of their effector functions. In support of this dual role of a costimulatory molecule, a recently published study showed that the use of agonistic anti-CD40 Ab initially increased the number of tumor-specific CD8+ T cells, and then it accelerated their deletion in a tumor model system (37). In addition, another group has reported that agonistic mAbs against CD40 can successfully control chronic autoimmune inflammatory processes (38). These studies and ours suggest that opposing effects can be elicited by strong T cell stimulation in vivo, and the use of agonistic Abs against costimulatory molecules may help us to reveal the complex functions of these molecules. The agonistic Ab against 4-1BB was able to inhibit two different EAE models, including the murine relapsing model, which is clinically relevant, suggesting that it is possible to preferentially delete activated autoreactive T cells. The use of agonistic Abs against costimulatory molecules may provide a novel strategy for treating T cell-mediated autoimmune diseases.
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2 Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of Pathology, University of Chicago, MC3083, Chicago, IL 60637. E-mail address: yfu{at}midway.uchicago.edu
3 Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; GVHD, graft-vs-host disease; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; AICD, activation-induced cell death; DLN, draining lymph node; DTH, delayed-type hypersensitivity; 7-AAD, 7-aminoactinomycin D; PLP, proteolipoprotein.
4 R. A. Wilcox, D. B. Flies, G. Zhu, A. J. Johnson, K. Tamada, A. I. Chapoval, S. E. Strome, L. R. Pease, and L. Chen. Treatment of poorly immunogenic established tumors by harnessing ignorant cytotoxic T cells with epitope peptide and anti-4-1BB costimulatory monoclonal antibody. Submitted for publication.
Received for publication April 20, 2001. Accepted for publication November 2, 2001.
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