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* Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030; and Departments of Microbiology and
Toxicology, Oregon State University, Corvallis, OR 97331
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, IL-6, and IFN-
, nor is
it dependent on the transcription factor NF-
B. However, in vivo
proliferation of NF-B-deficient T cells was comparable to that of
wild-type T cells, yet their early accumulation in the lymph nodes was
severely reduced unless the mice were treated with LPS and an agonistic
CD40 mAb. Most importantly, we found that activation of two different
costimulatory signals, CD40 and OX40, could not substitute for LPS in
rescuing T cells from peripheral deletion. Perhaps surprisingly, these
data show that LPS delivers a qualitatively different signal than
multiple costimulatory signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the two-signal hypothesis effectively describes T cell activation, it does not fully address how long-lived memory T cells develop and stay alive. It is certain that memory T cell responses can develop after two-signal stimulation (8, 12), but it is not yet clear whether these responses are optimal or what factors direct T cells into a surviving state. A variety of poorly understood compounds known as adjuvants can enhance an immune response and may be central to uncovering how T cells survive.
LPS, a natural adjuvant, is well known for having proinflammatory
properties. LPS is a component of Gram-negative bacterial cell walls
that induces potent inflammatory responses (13). Binding
of LPS to CD14 or Toll-like receptors on cells triggers the secretion
of reactive oxygen species and inflammatory cytokines such as TNF-,
IL-6, and IL-1 (14, 15). Additionally, LPS can activate
the transcription factor NF-B, a molecule that is well documented in
its ability to trigger cellular proliferation and cytokine secretion
among many other effects (16, 17).
LPS was previously shown to promote the long-term survival of T cells
stimulated with the superantigen
(SAg)3 staphylococcal
enterotoxin A (SEA) or with nominal peptide Ags (8, 18).
This survival effect could develop in the absence of CD28 signaling and
was at least partially dependent or partially substituted by
proinflammatory cytokines like TNF-, IL-1, and IFN-
(19).
Subsequent work with LPS found that, although it promoted significant levels of T cell survival, its effects were synergistic when coupled with OX40 costimulation in the presence of either SEA or OVA as the Ag (8). Delivery of these three signals not only enhanced T cell expansion compared with delivery of two signals in the form of Ag and costimulation, but it significantly enhanced the number of T cells expressing a memory phenotype for at least 2 mo. Thus, delivery of a natural adjuvant like LPS in the context of the two-signal model can potentiate T cell clonal expansion, but even more importantly, it can drive the long-term survival of those responding cells.
In this paper, we sought to better understand the mechanism of how LPS
was working in a three-signal model of T cell stimulation involving TCR
ligation (signal 1), CD40 stimulation (signal 2), and LPS (signal 3).
SAgs like SEA have proven very useful when studying clonal expansion
and tolerance of T cells in vivo (20, 21). Their
specificity for T cells possessing particular variable -chains
provides a reliable method for tracking T cells responding to the SAg
in vivo (22). CD40 is a member of the TNFR family that is
found primarily on APCs (23, 24, 25) and that upon stimulation
can enhance the activation and survival of APCs during an immune
response (26, 27, 28). SEA and CD40 stimulation together
enhance T cell clonal expansion in a CD28-dependent manner, but this is
followed by profound deletion (7).
We show that LPS stimulation in conjunction with SEA and CD40
stimulation markedly enhanced T cell survival in vivo. Interestingly,
the survival effect was not a result of enhanced costimulation, because
combined CD40 and OX40 costimulatory signals could not substitute for
LPS. We found that LPS-dependent T cell survival did not rely on
proinflammatory cytokines such as TNF-, IL-1, IL-6, and IFN-.
Moreover, LPS-independent short-term T cell survival was NF-B
dependent, but inclusion of LPS could rescue T cells in the short and
long term in the absence of NF-B. The results presented in this
paper begin to define a third signal, which cannot be substituted for
by two different costimulatory signals and is NF-B independent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.A mice were purchased from the National Cancer Institute
(Frederick, MD). C57BL/6, TNFRI knockout (KO), TNFRII KO, TNFRI/II
double KO (29), and B6, D2-TgN(LCK-NFKBIA)5Dwb mice
(30) (hereafter referred to as inhibitory protein that
dissociates from NF-B transgenic (IB- Tg) mice)
were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice
were maintained in our animal facility at Oregon State University under
specific pathogen-free conditions in accordance with federal
guidelines. All mice were between 6 and 12 wk of age.
Reagents, mAbs, and flow cytometry
SEA, staphylococcal enterotoxin B (SEB), LPS, and rat IgG were purchased from Sigma-Aldrich (St. Louis, MO) and administered to mice as i.p. injections in balanced salt solution (BSS) or PBS. The recombinant human IL-1Ra was a kind gift from Amgen (Boulder, CO).
The anti-CD40 mAb-producing hybridoma FGK45.5 was a kind gift from
Dr. A. Rolink (Basel Institute, Basel, Switzerland)
(31). The anti-IFN--producing hybridoma XMG1.2
(32), the anti-TNF--producing hybridoma MP6-XT22
(33), and the anti-IL-6-producing hybridoma
MP5-20F3.11 (34) were all obtained from the American Type
Culture Collection (Manassas, VA) with anti-IL-6 being obtained with
permission from DNAX (Palo Alto, CA). Supernatants from each of the
above hybridomas were purified over protein G agarose (Life
Technologies, Grand Island, NY) and dialyzed against PBS for
injection.
For flow cytometric staining, Abs purchased from BD PharMingen (San
Diego, CA) were used: anti-CD4 was conjugated to either PE or APC,
anti-CD8 was conjugated to either PE or APC, and anti-TCR V8
was conjugated to FITC. The anti-TCR V3 mAb KJ25-607.7
(35) was purified from hybridoma supernatant over protein
G agarose (Life Technologies) and conjugated to FITC as described
previously (36). Briefly, purified Ab was dialyzed against
0.1 M NaHCO3, pH 9.4–9.6. Protein concentration
was adjusted to 1 mg/ml and incubated with FITC-Celite (Sigma-Aldrich)
for 30 min at room temperature. Free celite was removed by
centrifugation and the Ab was dialyzed against PBS for use.
Injection schedule
All injections were i.p. The injection of the SAgs SEA and SEB
was considered day 0. Injection of anti-CD40 mAb was done 2 days
before SAg injection. LPS was always injected 24 h after the SAgs
(day +1). The anti-OX40 mAb was injected 24 and 36 h after SAg
injection. For the cytokine blocking experiments in Fig. 5
, b and c, we used the neutralizing mAbs
anti-TNF-, anti-IL-6, and anti-IFN-, as well as
IL-1Ra. These reagents were injected 20 and 30 h after
SEA.
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Spleens and peripheral lymph nodes (inguinal, axillary, and bronchial) were teased through nylon mesh sieves (Falcon, BD PharMingen) and RBCs were lysed with ammonium chloride. After washing, cells were counted with a Z1 particle counter (Beckman Coulter, Miami, FL), and spleen cells were further purified over nylon wool as described previously (37). Briefly, 3-ml syringes were filled with 0.12–0.15 g of washed and brushed nylon wool. The columns were prepared with warm BSS 5% FBS, after which the cells were loaded in a 0.5-ml volume and incubated for 30 min at 37°C. After draining 0.5 ml away, the columns were incubated for an additional 30 min, followed by elution with BSS 5% FBS.
For two- and three-color staining, cells were incubated on ice with the
primary Abs in the presence of 5% normal mouse serum, culture
supernatant from hybridoma cells producing an anti-mouse FcR mAb,
2.4.G2 (38), and 10 µg/ml human -globulin
(Sigma-Aldrich) to block nonspecific binding. After a 30-min incubation
on ice in staining buffer (BSS, 3% FBS, 0.1% sodium azide) with
primary Abs, the cells were washed twice and analyzed by flow
cytometry, or if a secondary reagent was necessary, the incubation and
wash procedures were repeated. Flow cytometry was conducted on a
FACSCaliber flow cytometer (BD Biosciences, Mountain View, CA), and the
data were analyzed using CellQuest software (BD PharMingen).
Bromodeoxyuridine (BrdU) staining
Mice were injected with 60 µg of SEB, 0.20 mg of
anti-CD40, and 10 µg of LPS at the times described above.
Additionally, the mice were injected with 1 mg of BrdU (Sigma-Aldrich)
dissolved in PBS on days 0, 1, and 2. On day 3, T cells from the
peripheral lymph nodes (LNs) and spleens from the treated mice were
isolated and stained with biotin-conjugated anti-TCR V8 mAb and
then with PE-conjugated streptavidin (BD PharMingen). The cells were
then stained with a modified BrdU staining protocol (39).
Briefly, the cells were dehydrated and fixed in ice-cold 95% ethanol
and then were fixed in BSS containing 1% paraformaldehyde and 0.01%
Tween 20. Next, cellular DNA was lightly digested with 50 Kunitz U of
DNase I (Sigma-Aldrich), and the cells were stained with
anti-BrdU-FITC (BD PharMingen).
In vitro proliferation
Mice were injected with 0.30 µg of SEA, 0.25 mg of anti-CD40 mAb, and 10 µg of LPS as described above. Ten days after SEA injection, spleen cells were isolated for culture. Cells from each in vivo treatment group were plated in triplicate at 5.0 x 105, 2.5 x 105, 1.25 x 105, and 0.63 x 105 cells per well in complete tumor medium (CTM). CTM consists of minimal essential medium with FBS, amino acids, salts, and antibiotics. SEA was added to the wells at a concentration of 1 µg/ml. The cultures were left for 72 h, with 1 µCi of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) being added for the last 8 h. Incorporation of [3H]thymidine was measured on a 1450 Microbeta Trilux Scintillation Counter (Wallac, Turku, Finland).
RT-PCR
B10.A mice were injected with 0.25 mg of anti-CD40, 0.30
µg of SEA, and 10 µg of LPS as described. At the time points
corresponding to 0.5, 1.5, and 6 h after LPS injection (or 24.5,
25.5, and 30 h after SEA injection), peripheral LNs and spleens
were removed, and total RNA was prepared from the cell suspensions
using RNAwiz according to the manufacturer’s suggested protocol
(Ambion, Austin, TX). Synthesis of cDNA was performed with 1 µg of
RNA using the Reverse Transcriptase System (Promega, Madison, WI) and
accompanying protocol. Aliquots of cDNA were amplified with the GeneAmp
PCR System 2400 (Perkin-Elmer, Norwalk, CT). Amplification conditions
consisted of a 4-min denaturation step at 94°C, followed by 30 cycles
of amplification (95°C for 1 min, 53°C for 1 min, 72°C for 1 min)
and a final extension at 72°C for 7 min. PCR products were
electrophoretically separated on a 2% agarose gel containing ethidium
bromide. The polycompetitor plasmid PQRS (40) was a
kind gift from Dr. Reiner (University of Chicago). This plasmid
contains cDNA sequences for each cytokine amplified and was used as a
positive control. The sequences of the primers are as follows:
hypoxanthine phosphoribosyltransferase, 5'-GTT GGA TAC AGG CCA GAC TTT
GTT G-3' and 5'-GAG GGT AGG CTG GCC TAT AGG CT-3'; TNF-, 5'-GTT CTA
TGG CCC AGA CCC TCA CA-3' and 5'-TAC CAG GGT TTG AGC TCA GC-3';
IL-1, 5'-AAG CTC TCC ACC TCA ATG GAC AG-3' and 5'-CTC AAA CTC CAC
TTT GCT CTT GA-3'; IL-2, 5'-TCC ACT TCA AGC TCT ACA G-3' and 5'-GAG TCA
AAT CCA GAA CAT GCC-3'; IL-6, 5'-CCT CTG GTC TTC TGG AGT ACC AT-3' and
5'-GGC ATA ACG CAC TAG GTT TGC CG-3'; IL-15, 5'-ACT GAC AGT GAC TTT CAT
CCC A-3' and 5'-GTG CTG CCT CTG AGC AGC AGG-3'; and IFN-, 5'-CAT TGA
AAG CCT AGA AAG TCT G-3' and 5'-CTC ATG AAT GCA TCC TTT TTC G-3'. All
primers were made by the Central Services Laboratory at Oregon State
University (Corvallis, OR).
Subcellular fractionation
Combined LN and spleen cells from C57BL/6 and IB- Tg mice
were cultured overnight at a concentration of 10 x
106 cells/ml in CTM in the presence of 2 µg/ml
Con A. T cells were purified over nylon wool to between 50 and 90%
purity.
Nuclear and cytoplasmic extracts were prepared as described (41). Briefly, cell pellets were resuspended in sucrose buffer (0.32 M sucrose, 3 mM CaCl2, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 2 mM Mg acetate, 1 mM DTT, 0.5 mM PMSF) with 0.5% (v/v) IGEPAL by gentle pipetting and were centrifuged. To the cytoplasmic fraction, 0.22 volumes of 5x cytoplasmic extraction buffer (0.15 M HEPES, 0.7 M KCl, 0.015 M MgCl2) was added. The cytoplasmic fraction was then centrifuged at 15,000 rpm in a microcentrifuge, and the supernatant was transferred to a fresh tube containing 25% (v/v) glycerol and stored at -80°C. The nuclei were washed twice in sucrose buffer without IGEPAL. Nuclei were then resuspended in low salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), and then 1 volume of high salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM KCl, 0.2 mM EDTA, 1% IGEPAL, 0.5 mM DTT, 0.5 mM PMSF) was carefully added in 1/4 increments. Nuclei were incubated on ice for 30 min, diluted 1/2.5 with diluent (25 mM HEPES, 25% glycerol, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), and centrifuged at 15,000 rpm in a microcentrifuge at 4°C. Nuclear lysates were stored at -80°C.
DNA binding assay
EMSAs were used to assess sequence-specific binding of DC2.4
nuclear NF-B/Rel to DNA (41). Briefly, a synthetic
20-bp consensus B response element probe (upper strand,
5'-GAT CGG CAG GGG AAT TCC CC-3'; and lower strand, 5'-GAT CGG GGA ATT
CCC CTG CC-3') was labeled with [-32P]dATP
using Klenow fragment (Invitrogen, Carlsbad, CA) and used for DNA
binding assays. Nuclear extracts were prepared as described above.
Samples (5 µg) were incubated with binding buffer (12 mM HEPES, pH
7.3; 4 mM Tris-HCl, pH 7.5; 100 mM KCl; 1 mM EDTA; 20 mM DTT; 1 mg/ml
BSA), 4 µg of poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway,
NJ), and 100,000 cpm of 32P-labeled B response
element for 20 min at room temperature. Anti-RelA,
anti-RelB, anti-c-rel, anti-p50, and
anti-p52 were added to the reaction mixture according to
manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA)
and incubated for 10 min at room temperature. Samples were analyzed on
a 5% polyacrylamide gel in 0.5% Tris-buffered EDTA (44.5 mM Tris,
44.5 mM boric acid, 1 mM EDTA) and visualized by autoradiography.
Immunoblotting
Cytoplasmic extracts were subjected to SDS-PAGE as described
(42). Proteins were transferred to nitrocellulose
membranes (Bio-Rad, Hercules, CA) in 25 mM Tris (pH 8.3), 192 mM
glycine, 20% methanol using a Genie Electroblotter (Idea Scientific,
Minneapolis, MN). Membranes were blocked overnight at 4°C in TBS (25
mM Tris, pH 7.4, 150 mM NaCl) containing 5% nonfat dry milk. Abs were
diluted in TBS containing 1% nonfat dry milk and the membranes were
incubated with primary Abs for at least 1.5 h at room temperature.
Anti-IB- IgG (Santa Cruz Biotechnology) and HRP-conjugated
secondary Abs, donkey anti-rabbit IgG (Amersham Pharmacia Biotech)
were used according to the manufacturer’s instructions. After each Ab
treatment, blots were washed in TBS containing 0.05% Tween 20. Ab
complexes were visualized with chemiluminescent reagents (Pierce,
Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous work with CD40 stimulation found that agonistic anti-CD40 mAb treatment could enhance SEA-mediated T cell expansion in vivo; however, it could not keep the responding T cells alive for very long (7). After 2–3 wk, Ag-specific T cell numbers declined to levels observed in mice treated with SEA alone. This death process was not prevented, only delayed.
A very similar trend was observed during treatment with an anti-OX40 agonistic mAb (8). OX40 stimulation enhanced Ag-specific T cell expansion and, much like CD40 stimulation, could only yield weak long-term survival. However, injection of LPS into mice treated with both Ag and anti-OX40 mAb strongly enhanced T cell survival beyond 2 mo.
With these observations, we hypothesized that LPS injection would
prevent the death of T cells from mice treated with SEA and
anti-CD40 mAb. Thus, mice were injected with the anti-CD40
agonistic mAb, SEA, and LPS. At various time points after Ag treatment,
T cell populations were examined in the lymphoid tissues. Fig. 1 shows a time course of the clonal
expansion and survival of splenic CD4 V3 (Fig. 1, a and
c) and CD8 V3 (Fig. 1, b and d) T
cells stimulated by a combination of SEA, anti-CD40 mAb, and
LPS.
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3 T cell percentages (Fig. 1
a) and numbers (Fig. 1c) declined on day 5 below
their starting levels. The peak of clonal expansion that normally
occurs on day 2–3 was not examined. Co-injection of either
anti-CD40 mAb or LPS with SEA produced increased percentages and
numbers of T cells on day 5, with LPS yielding more potent T cell
expansion and short-term survival than CD40 stimulation. By day 14,
however, T cells from these SEA/anti-CD40 or SEA/LPS-treated mice
had slightly increased numbers of V3 T cells compared with those
mice treated with SEA alone (Fig. 1, c and d).
Based on percentages of CD4 V3 T cells on day 14, mice treated with
SEA/LPS had 1.5-fold more splenic Ag-specific T cells (3.40 ±
0.49%), and SEA/anti-CD40-treated mice had 2-fold more splenic
Ag-specific T cells (4.70 ± 0.25%) than SEA-treated mice
(2.31 ± 0.12%). Similar differences were observed when examining
T cell numbers (Fig. 1c). Thus, a low level of T cell
survival is produced with SEA/anti-CD40 or SEA/LPS treatment.
What is most striking about the results is that a combination of the
three signals (SEA, anti-CD40 mAb, and LPS) prevented many more T
cells from deleting. This effect is observed by examining both
percentages and numbers. V3 T cells from mice stimulated by the
three signals accumulated almost 10-fold from their starting
populations by day 5 and had declined slightly in number after 2 wk. On
day 14, CD4 V3 percentages were increased by 16-fold over SEA alone
to 38.46 ± 3.08% (Fig. 1a), whereas the absolute
numbers were increased by nearly 13-fold to 40.27 x
105 ± 2.69 (Fig. 1c).
A very similar response to that described above was observed in the LN
(data not shown) as well as in the Ag-specific CD8 T cell population
(Fig. 1, b and d). In mice injected with SEA,
anti-CD40 mAb, and LPS, splenic CD8 V3 T cells accumulated to a
much greater level than any other treatment on day 5 and then began to
gradually decline in both percentage (Fig. 1b) and number
(Fig. 1d). By day 14, there was a 26-fold increase in the
percentage of CD8 V3 T cells in three-signal-treated mice
(29.90 ± 4.86%) vs SEA-treated mice (1.13 ± 0.02%). In
contrast, SEA/LPS- and SEA/anti-CD40-treated mice only produced a
4-fold increase in Ag-specific T cell accumulation. Thus, both
Ag-specific CD4 and CD8 T cell populations can be signaled to survive
beyond the effector stage by the combined action of signal 1 (SAg),
signal 2 (costimulation), and signal 3 (LPS).
We next tested whether these rescued T cells were functional by
stimulating them in vitro with recall Ag using SEA. As shown in Fig. 2, mice treated with three signals in
vivo for 10 days proliferated effectively when restimulated in vitro.
This proliferation was less on a per cell basis than other weaker in
vivo treatments yielded in vitro, which was likely due to the depleted
resources and energetic costs of undergoing such a potent response.
These data show that the T cells that are rescued from death by the
combination of SAg, costimulation, and LPS are not anergic.
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Stimulation of APCs through CD40 causes an increase in the expression of both B7 molecules as well as MHC class II (7, 43). In addition, LPS stimulation can also enhance B7 expression and prolong OX40 expression on activated T cells (8, 44). We hypothesized that because LPS can synergize with both CD40 and OX40 stimulation to promote Ag-specific T cell survival, its mechanism of action may involve combined signaling via CD28 and OX40 on the SAg-specific cells.
To test this idea, we injected mice with a triple combination of SEA,
an agonistic anti-CD40 mAb, and an agonistic anti-OX40 mAb. The
time course of this experiment is shown in Fig. 3, a and b, and the
same scale as in Fig. 1 is used to allow a direct comparison between
the two figures.
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a) and CD8 (Fig. 3b) T cells expressing V3 behaved in a similar manner.
Treatment of mice with SEA and anti-OX40 mAb enhanced the
percentage of Ag-specific T cells in the spleen above that observed
with SEA alone on day 3, but on subsequent days, the difference between
the two treatments was minimal. SEA and anti-CD40 mAb treatment
enhanced the CD4 V3 and CD8 V3 percentages in the spleen at the
peak of expansion by 4- to 5-fold above that observed with SEA alone.
By days 7 and 12, however, there was less than a 2-fold increase in the
percentage of T cells remaining. When SEA, anti-CD40 mAb, and
anti-OX40 mAb were injected in combination, the observed response
was very similar to that from injecting SEA and anti-CD40 mAb.
Ag-specific T cell expansion was enhanced 4- to 5-fold above starting
levels on day 3, but after day 3, little survival was observed. There
was a slightly larger percentage of Ag-specific T cells remaining on
day 12 after the combined treatment (
4-fold above SEA alone), but
this was a small fraction of that observed with LPS (Fig. 1). Thus,
costimulation via CD40 and OX40 can yield small levels of T cell
survival, but when combined with signals produced upon LPS injection,
the survival effect is greatly potentiated, suggesting that LPS does
not rescue Ag-specific T cells merely by stimulating through OX40
or CD40. Because the survival response observed during three-signal stimulation does not appear to be a sum of multiple costimulatory signals being delivered, the next focus of study was whether the LPS response could be enhanced further. Because LPS can synergize with either CD40 or OX40 stimulation to improve Ag-specific T cell survival, it was necessary to examine whether combining all of these signals could enhance the amount of surviving T cells.
Mice were injected with a combination of SEA, LPS, anti-CD40, and
anti-OX40. After 10 days, the T cell populations were examined. In
mice injected with SEA, anti-CD40, and LPS, 18.96 ± 2.86% of
the CD4 T cells (Fig. 3c) and 8.54 ± 1.49% of the CD8
T cells (Fig. 3d) were SEA specific. A slightly larger
percentage was observed in mice injected with SEA, anti-OX40, and
LPS: 19.50 ± 3.81% CD4 V3 and 9.26 ± 2.23% CD8 V3 T
cells. In mice treated with both mAbs, SEA and LPS, little enhancement
of T cell survival was observed, increasing to 24.29 ± 4.27% in
the CD4 V3 population and 11.56 ± 3.17% in the CD8 V3
population. In these experiments, we used 2.5 times less anti-CD40
mAb to conserve on reagent, and thus the magnitude of the response is
lessened compared with Fig. 1. Increasing the LPS dose to 15 µg did
not further enhance T cell survival, yielding slightly lower
percentages and numbers of SEA-specific T cells than observed with 10
µg (data not shown). Again, the numbers of SEA-specific T cells as
well as the LN cells both behaved in a similar fashion (data not
shown). Thus, costimulation of T cells via either CD40 or OX40
stimulation in the presence of LPS can produce a substantial population
of surviving Ag-specific T cells that cannot be significantly augmented
by additional costimulation through these receptors. This again
suggests that single or multiple costimulatory signals are not the
major factor determining T cell survival beyond the effector phase
in vivo.
Proinflammatory cytokines are not necessary for T cell survival in the presence of anti-CD40 stimulation and LPS
LPS stimulation of APCs can trigger the production of many
proinflammatory cytokines. To begin to understand how LPS promoted
survival in our model, mRNA expression of various cytokines was
examined at early time points after LPS injection by RT-PCR (Fig. 4). The goal was to identify candidate
cytokines that may be contributing to the survival response.
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were enhanced upon LPS treatment in the LN and spleen. All
reactions were normalized to hypoxanthine phosphoribosyltransferase,
which allowed for semiquantitative comparisons to be made. TNF-,
IL-1, IL-6, IL-15, and IFN- mRNA were all up-regulated to varying
degrees during the first 6 h after LPS treatment compared with
mice that received no LPS but were examined at the exact same time.
IL-2 was also examined, but it was not observed in any treated or
untreated mouse at the times tested (data not shown).
Previous work with mice injected with SEA and LPS showed that TNF-
can substitute to a small degree for LPS in keeping Ag-specific T cells
alive (19). The survival effect was not nearly as potent
as that observed with LPS injection, but it was significant. This
result, coupled with the fact that TNF- message expression was
increased by LPS injection in the three-signal model system, suggested
that TNF- might be at least partially responsible for long-term T
cell survival in the model described in this report.
To investigate the importance of this cytokine in the three-signal
model system, mice deficient in either or both TNFRs were used
(29). Because the knockout mice are on a C57BL/6
background, we used SEB instead of SEA to avoid the effect of
endogenous mouse mammary tumor viruses on V3 T cells. Each mouse
strain was injected with SEB and anti-CD40 mAb with or without LPS,
and lymphoid tissues were examined on day 10 for the presence of
SEB-specific T cells (TCR V8). Fig. 5a shows the percentage of CD4
V8 T cells in the spleens of the treated mice 10 days after SEB
injection.
In C57BL/6 mice treated with SEB and anti-CD40 stimulation, CD4
V8 T cells declined to an average percentage of 18.00 ± 0.92%
after 10 days. When those mice were given LPS, the percentage of
SEB-specific T cells rose to 32.69 ± 2.85%; an 85% increase.
Mice deficient in TNFRI had 22.30 ± 1.18% splenic CD4 V8 T
cells in the absence of LPS treatment, but a 63% increase to
36.34 ± 2.13% was observed when LPS was co-injected. T cells
from TNFRII knockout mice increased by 69% upon LPS treatment, rising
from 18.35 ± 0.79% CD4 V8 T cells without LPS to 31.13
± 3.30% with LPS.
In the absence of one TNFR, there remained the possibility that the
other receptor was still delivering a survival signal due to redundancy
of function between the two receptors. Thus, mice deficient in both
TNFRs were treated with three signals as in the other strains. Mice
receiving SEB and anti-CD40 mAb had a CD4 V8 T cell percentage
on day 10 of 20.03 ± 1.00%, but when injected with LPS, the
percentage rose 63% to 32.65 ± 1.79%, similar to that observed
in the other mice. Overall, these data suggest that signaling through
TNFRI and TNFRII is dispensable for long-term T cell survival when CD40
stimulation is used in combination with LPS.
The observation that T cell survival can still occur in the absence of TNFR signaling does not mean that such signaling cannot contribute to survival. Other proinflammatory cytokines may be contributing a survival signal and thus may be substituting for TNFR signaling. To get at the issue of whether other cytokines were delivering the signals necessary to keep activated T cells alive, a combination of cytokines was blocked in vivo during SEA, anti-CD40, and LPS stimulation.
B10.A mice were injected with SEA, anti-CD40, and LPS, but before
and after LPS injection, IL-1Ra, anti-TNF- mAb and anti-IL-6
mAb were all injected to try to neutralize as much activity of these
three cytokines as possible. Because the RT-PCR data showed that
IFN- expression was also increased after LPS injection (Fig. 4),
another treatment group was set up in which IFN- was blocked with a
mAb to investigate what effect this cytokine had on survival. The
percentages (Fig. 5b) and numbers (Fig. 5c) of
CD4 V3 T cells 10 days after SEA injection are shown.
SEA treatment led to the expected decline in T cell percentages,
decreasing from a 6% CD4 V3 T cell population in uninjected mice to
a little over 2% by day 10. SEA/anti-CD40 mAb and SEA/LPS
treatments both produced small increases in T cell percentages in
comparison to SEA alone, again showing that two signals can generate
some T cell survival post effector stage. Mice treated with
SEA/anti-CD40/LPS and the control IgG exhibited a large increase in
percentage of SEA-specific T cells to 22.12 ± 4.32%. What was
most interesting, however, was that when IL-1, TNF-, and IL-6
were all neutralized, the percentage of surviving T cells actually
increased slightly to 27.42 ± 5.34%. Even blocking IFN- did
not decrease survival, yielding 23.25 ± 6.56% CD4 V3 T cells
after 10 days in vivo.
A much better enhancement of T cell survival after cytokine
neutralization was observed based on CD4 V3 T cell numbers (Fig. 5c). Mice receiving three-signal stimulation and the control
rat IgG had 23.28 x 105 ± 6.31
SEA-specific T cells remaining after 10 days. Blocking TNF-,
IL-1, and IL-6 increased the numbers of surviving T cells to
42.40 x 105 ± 11.2, whereas IFN-
inhibition increased the numbers to 35.83 x
105 ± 14.62.
CD8 V3 T cells responded a little differently to the cytokine
blocking studies than the CD4 V3 T cells did (data not shown). Both
percentages and numbers of CD8 T cells expressing V3 in the spleen
were increased poorly, if at all, by inhibition of proinflammatory
cytokines. This could represent a difference in activation/survival
requirements between CD4 and CD8 T cells. However, what is identical
between both kinds of T cells is that blocking TNF-, IL-1, IL-6,
and IFN- did not adversely affect survival and, perhaps
surprisingly, enhanced T cell survival to varying degrees.
To verify that the cytokines were inhibited by the neutralizing mAbs,
serum samples were taken from the injected mice 1 day after the LPS and
Ab injections. ELISAs were performed using anti-rat IgG to detect
the neutralizing Abs in the serum using a sandwich ELISA. In the
control rat IgG group, quantities of rat IgG were detected in the serum
that exceeded the maximum detectable amount (data not shown). In the
anti-TNF--, anti-IL-6-, and IL-1Ra-treated groups there was
slightly less Ab detected, but still large amounts remained in the
serum (data not shown). The anti-IFN--treated mice also had
detectable levels of rat IgG in their serum (data not shown). The ELISA
data show that there were large amounts of rat IgG Abs present in the
serum over 24 h after LPS treatment. This suggests that the
cytokines were neutralized soon after they were secreted, and thus this
data is consistent with the data collected from the TNFR KO mice.
Furthermore, attempts to substitute LPS with TNF-, IL-1, and IL-6
to induce SAg-specific T cell survival failed, even though large doses
of the cytokines were used (data not shown). Collectively, these data
suggest that CD40 stimulation circumvents a role for proinflammatory
cytokines in long-term T cell survival.
Similar results were observed when IL-1 and IL-6 were inhibited in
TNFR double-knockout mice (data not shown). No adverse effects on
long-term T cell survival were observed in this model system, and some
slight increases in surviving T cell numbers were observed. Thus, from
these data it appears that the proinflammatory cytokines TNF-,
IL-1, IL-6, and IFN- are not necessary for long-term T cell
survival and in fact may actually be somewhat detrimental, possibly
playing a role in attenuating an Ag-specific CD4 T cell response to Ag,
strong costimulation, and LPS treatment. This is consistent with
previous reports showing that TNF- and IFN- readily exert
cytotoxic effects on lymphocytes (40, 45, 46). IL-1 and
IL-6, however, appear to be more protective (47, 48),
although IL- does play a role in the death of -cells in diabetes
(49).
A mixed role for NF-B during T cell survival
The transcription factor NF-B is very important for
inflammatory responses as well as T cell proliferation and survival
(16, 17, 50, 51, 52). Many inflammatory cytokines, as well as
LPS, activate transcriptional activity of this molecule
(53, 54, 55, 56). To examine the role of NF-B in T cell
survival in vivo, mice Tg for a mutant IB- molecule that cannot
be degraded were used (30). IB- is a repressor of
NF-B that prevents the translocation of NF-B into the nucleus
(57). When IB- is phosphorylated, it is targeted for
ubiquitination (58, 59). Subsequent degradation of
IB- releases NF-B and allows it to translocate to the nucleus
and initiate transcription. Thus, these mutant IB- Tg mice have
reduced NF-B binding to DNA response elements. Additionally, this
transgene is under the control of the lck promoter, thus limiting its
expression, and ultimately NF-B inhibition, to the T cell
population, allowing an examination of how effective T cell survival is
in the presence of reduced NF-B activity.
As shown in Fig. 6a, the
percentage of splenic CD4 V8 T cells in C57BL/6 mice given SEB and
anti-CD40 mAb without LPS was 20.91 ± 0.83% and rose to
33.26 ± 3.35% when LPS was injected. A very similar increase was
observed in the IB- Tg mice whose T cell populations increased
from 22.97 ± 1.12% without LPS to 33.90 ± 3.03% with LPS
(Fig. 6b). These numbers correspond to a 59% difference
between treatments in C57BL/6 mice and a 48% difference in the Tg
mice. A similar trend was observed in the absolute numbers of CD4 V8
T cells, which increased by 32% in the C57BL/6 mice and by 31% in the
Tg mice that received LPS (data not shown). A few individual mice had
much higher levels of CD4 V8 T cells than observed in control
strains. One Tg mouse in particular had over 70% of its CD4 T cells
bearing V8 in the spleen 10 days after SEB injection. This is in
contrast to C57BL/6 mice, which never rose above 50%.
|
B- by Western blot and EMSAs assessing NF-B
activity were performed using T cells from mice shown in Fig. 6b. The percentages shown in the lanes of the Western and
EMSA blots represent the percentage of CD4 V8 T cells in
SEB/anti-CD40/LPS-treated mice used in Fig. 6b. Thus,
the 39% lane represents cells from a mouse that had 39% CD4 V8 T
cells in Fig. 6b. The data show little variation in
transgene expression regardless of the amount of rescue (Fig. 6c). Furthermore, data measuring NF-B activity showed
profound inhibition of NF-B in the cells taken from the transgenic
mice as expected (Fig. 6b). Therefore, these controls
support our hypothesis that survival of Ag-activated T cells beyond the
effector stage is not dependent on NF-B.
LPS can circumvent proliferation defects in NF-B-deficient T
cells
Because LPS-induced long-term T cell survival can still occur in
NF-B-deficient T cells, it was important to investigate whether T
cell responses were deficient and whether LPS could somehow overcome
that deficiency. Thus, IB- Tg or C57BL/6 mice were injected with
different combinations of SEB, anti-CD40 mAb, and LPS and were
given BrdU injections for 3 days. After this treatment, the percentages
and numbers of Ag-specific T cells incorporating BrdU were evaluated
(Fig. 7).
|
B- Tg mice had similar percentages of V8 T
cells incorporating BrdU in the LN on day 3 (Fig. 7, a and
c). Based on these percentages of BrdU incorporation, no
major proliferation defect was apparent. SEB treatment mediated 40%
of the responding T cells to take up BrdU in both normal and IB-
Tg mice. Three-signal treatment only slightly enhanced the
incorporation of BrdU to 50% of the V8 population.
When the absolute numbers of V8 T cells taking up BrdU was examined,
however, a major defect was observed (Fig. 7, b and
d). C57BL/6 mice had similar numbers of V8 T cells
incorporating BrdU regardless of whether SEB alone or three signals
were delivered. The number of NF-B-deficient T cells taking up BrdU
was strongly reduced in SEB-treated mice, strongly supporting the
notion that NF-B activity promotes survival during clonal expansion.
However, co-injection of anti-CD40 and LPS with SEB increased the
numbers by 4.5-fold, suggesting that such stimulation enhances
short-term survival, as measured by accumulation of Ag-specific T
cells, in an NF-B-independent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b, shows that combined signaling
via CD40 and OX40 cannot yield the same level of survival as observed
in SEA/anti-CD40/LPS-treated mice. The percentages of SEA-specific
T cells observed in Fig. 3, a and b, are
increased 3- to 5-fold above SEA-treated mice when both costimulatory
signals are delivered, a response similar to what is observed when only
two signals are given (Fig. 1). Also, we found that simultaneous
stimulation of both CD40 and OX40 did not significantly enhance the LPS
response (Fig. 3, c and d), suggesting that under
these circumstances, when analyzing T cell survival, the CD40 and
OX40 signal is redundant or overlapping on a cellular level. It is
likely that LPS induces other kinds of responses that OX40 and CD40
stimulation cannot. Thus, optimal T cell survival occurs when three
qualitatively different signals are delivered to the T cell. We
conclude that delivery of signals 1, 2, and 3 is not the same as the
delivery of signal 1 and two signal 2s (Fig. 8).
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, IL-1, IL-6, and IFN- (Fig. 4). TNF-
was the first cytokine studied because previous work found that
blocking TNF- signaling in vivo with a mAb inhibited T cell survival
in response to SEA/LPS injection (18). Subsequent work
found that injection of TNF- in place of LPS promoted some T cell
survival after SEA injection (19). However, the percentage
of SEA-specific T cells surviving after SEA/TNF- treatment was only
about one-third of that observed with injection of SEA and LPS. The
fact that TNF- or IL-1 could not completely substitute for LPS in
enhancing survival implied that other cytokines might also contribute
to the overall survival response. Thus, whereas some decrease in T cell
survival might be expected in the absence of TNFR signaling, it may not
be completely abolished.
What is most apparent and perhaps surprising about the TNFR KO mouse
data shown in Fig. 5a is that neither TNFR is required for
effective T cell survival resulting from three-signal stimulation. This
is different from the results from SEA/LPS alone-treated mice. It has
been shown that TNF- does have survival-inducing capabilities during
antigenic signaling (60) and is an important component of
LPS-mediated T cell survival after SEA stimulation (18).
Nevertheless, we show for the first time that a strong costimulatory
signal via CD40 can bypass the requirement for TNF-.
One possibility is that other cytokines may be substituting for TNF-
in the TNFR KO mice. Earlier work suggested that IL-1 and IFN-
could also contribute to the survival response (18, 19).
Thus, combinations of these proinflammatory cytokines were neutralized
in the presence of three-signal stimulation to test whether signals
from multiple cytokines delivered the survival stimulus.
TNF-, IL-1, and IL-6 were all neutralized in the presence of SEA,
anti-CD40, and LPS. Again, the inhibition of these three cytokines
did not block T cell survival, but slightly enhanced it in the
SEA-specific CD4 T cell population (Fig. 5, b and
c). Furthermore, the survival response was also unaffected
or slightly enhanced by a blockade of IFN-. Thus, there appears to
be no significant role for these important inflammatory cytokines in T
cell survival induced by three-signal stimulation.
Because the proinflammatory cytokines we tested were not involved in
survival in this model, we reasoned that analysis of transcription
factors may narrow our search for mediators of survival. NF-B is an
important molecule in T cell responses and is activated by signals from
LPS and many proinflammatory cytokines (50, 53).
Activation of NF-B has been shown by many laboratories to inhibit
apoptosis (54, 55, 56, 61). Additionally, NF-B is
responsible for transcribing many survival genes such as A1, A20, and
Bcl-xL (62, 63). Thus, we tested the
hypothesis that a deficiency in NF-B signaling would alter
three-signal-induced T cell survival by using Tg mice that express a T
cell-specific trans-dominant form of the NF-B repressor,
IB-, that cannot be degraded (30). These mice and
similar strains were shown to be deficient in T cell proliferation and
IL-2 secretion in vitro (64, 65, 66). Additionally, the T
cells were found to be more prone to apoptosis in vitro after
activation. The majority of these studies were conducted in vitro, and
very little is known about how these unusual peripheral T cells respond
in vivo.
Injecting the Tg mice with SEB, anti-CD40 mAb, and LPS continued to
produce effective survival that was comparable to that observed in
C57BL/6 mice (Fig. 6, a and b). It was shown in
this report that upon exposure to three signals, the NF-B-deficient
T cells can survive for long periods of time in vivo even though
activated NF-B-deficient T cells are readily susceptible to
apoptosis in vitro.
Although Western blots showed that IB- was expressed in
equivalent amounts in all Tg mice examined (Fig. 6c), it was
possible that this mutation did not completely inhibit NF-B
activity. Thus, the possibility remained that some small level of
transcription by NF-B was still occurring in the Tg cells that
transcribed the survival factor. To further test this idea, EMSAs were
performed on some of the T cells taken from the mice in Fig. 6b. Although there were very low and somewhat variable
levels of detectable NF-B DNA-binding activity in cells from the Tg
mice, no correlation was observed between NF-B activity and the
level of T cell survival in vivo. It is possible, however, that p50
homodimers are present in these cells at very low levels as previously
reported (30). Thus, NF-B does not appear to be
important for long-term survival of Ag-specific T cells in this
model.
The NF-B data are further supported by the cytokine data. In the
experiments where cytokines were neutralized, there presumably would be
less NF-B activation. Both TNF- and IL-1 activate NF-B, and
in the absence of signals through the receptors of these cytokines,
there should be less overall NF-B transcriptional activity. In this
situation, long-term T cell survival still occurred. Thus, the role of
NF-B in T cell survival appears to be dispensable.
A recent study from Mitchell et al. (67) suggested that
adjuvants such as LPS may be causing T cell survival by inducing Bcl-3,
which assists NF-B transcription. Such transcription was suggested
to produce important proteins responsible for survival. Our data
suggest that NF-B may not be necessary for long-term T cell
survival, but we cannot rule out the possibility that NF-B activity
can produce a survival factor. Because Bcl-3 plays a role in NF-B
transcription and T cell responses, it may be that NF-B is most
significant in the early phases of a T cell response for short-term
survival.
Proliferation, as measured by BrdU incorporation, was not affected
significantly by NF-B inhibition based on the percentage of T cells
incorporating BrdU (Fig. 7). However, when the absolute numbers of T
cells were examined, a dramatic decline in T cell numbers incorporating
BrdU was observed after SEB injection (Fig. 7). Thus, the SEB-specific
T cells divide normally, or at least attempt to divide normally, but
they do not effectively accumulate in the lymphoid tissues without
NF-B signaling.
We tested whether CD40 and LPS stimulation could circumvent the
defective accumulation of T cells in this response. Injection of these
three signals enhanced the numbers of SEB-specific T cells
incorporating BrdU on day 3 by 4-fold. Numbers were not restored to
levels observed in C57BL/6 mice, but they were enhanced, implying that
three-signal stimulation can override some of the inhibition of
short-term T cell survival that results from NF-B deficiency. These
data support the novel hypothesis that NF-B is important for
short-term survival of Ag-specific T cells as demonstrated by others
(67) but that nevertheless can be circumvented by combined
CD40 and LPS stimulation.
Although it is now clear that Ag is not necessary for maintenance of
memory CD4 or CD8 T cells (68, 69), the question that
remains is what external factors drive the decision of a cell to
develop into a memory cell vs one that dies? We previously have
proposed that three signals optimally induce the development of memory
T cells, and in this report we show that LPS and CD40 stimulation do
not obligatorily rely on proinflammatory cytokines as in other systems
(18, 19, 70). Collectively, these data show that CD40
stimulation can bypass the need for LPS-induced proinflammatory
cytokines, and they demonstrate that two different costimulatory
signals and Ag are not qualitatively the same as Ag, one costimulatory
signal, and a signal mediated by a natural adjuvant. These data lead us
to hypothesize that the third signal (LPS) drives long-term T cell
survival and is functionally different from a prototypical
costimulatory signal (Fig. 8).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anthony T. Vella, Department of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: vella{at}uchc.edu
3 Abbreviations used in this paper: SAg, superantigen; SEA, staphylococcal enterotoxin A; IB- Tg, inhibitory protein that dissociates from NF-B transgenic; SEB, staphylococcal enterotoxin B; BSS, balanced salt solution; BrdU, bromodeoxyuridine; LN, lymph node; CTM, complete tumor medium; KO, knockout.
Received for publication November 14, 2001. Accepted for publication February 15, 2002.
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  J. P. McAleer, R. J. Rossi, and A. T. Vella Lipopolysaccharide Potentiates Effector T Cell Accumulation into Nonlymphoid Tissues through TRIF J. Immunol., May 1, 2009; 182(9): 5322 - 5330. [Abstract] [Full Text] [PDF]
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J. P. McAleer, D. J. Zammit, L. Lefrancois, R. J. Rossi, and A. T. Vella The Lipopolysaccharide Adjuvant Effect on T Cells Relies on Nonoverlapping Contributions from the MyD88 Pathway and CD11c+ Cells J. Immunol., November 15, 2007; 179(10): 6524 - 6535. [Abstract] [Full Text] [PDF]
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J. Dai, B. Liu, S. M. Ngoi, S. Sun, A. T. Vella, and Z. Li TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells J. Immunol., March 1, 2007; 178(5): 3219 - 3225. [Abstract] [Full Text] [PDF]
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J. S. Levine, R. Subang, S. H. Nasr, S. Fournier, G. Lajoie, J. Wither, and J. Rauch Immunization with an Apoptotic Cell-Binding Protein Recapitulates the Nephritis and Sequential Autoantibody Emergence of Systemic Lupus Erythematosus J. Immunol., November 1, 2006; 177(9): 6504 - 6516. [Abstract] [Full Text] [PDF]
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B. S. Thompson, P. M. Chilton, J. R. Ward, J. T. Evans, and T. C. Mitchell The low-toxicity versions of LPS, MPL(R) adjuvant and RC529, are efficient adjuvants for CD4+ T cells J. Leukoc. Biol., December 1, 2005; 78(6): 1273 - 1280. [Abstract] [Full Text] [PDF]
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R. J. Rossi, G. Muralimohan, J. R. Maxwell, and A. T. Vella Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int. Immunol., December 1, 2004; 16(12): 1751 - 1760. [Abstract] [Full Text] [PDF]
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S.-J. Lee, L. Myers, G. Muralimohan, J. Dai, Y. Qiao, Z. Li, R. S. Mittler, and A. T. Vella 4-1BB and OX40 Dual Costimulation Synergistically Stimulate Primary Specific CD8 T Cells for Robust Effector Function J. Immunol., September 1, 2004; 173(5): 3002 - 3012. [Abstract] [Full Text] [PDF]
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J. R. Maxwell, R. J. Rossi, S. J. McSorley, and A. T. Vella T Cell Clonal Conditioning: A Phase Occurring Early after Antigen Presentation but before Clonal Expansion Is Impacted by Toll-Like Receptor Stimulation J. Immunol., January 1, 2004; 172(1): 248 - 259. [Abstract] [Full Text] [PDF]
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), SEA/anti-CD40 (
),
SEA/LPS (
), and SEA/anti-CD40/LPS (•). Mice were injected with
0.25 mg of anti-CD40, 0.30 µg of SEA, and 10 µg of LPS. On days
5 and 14 after SEA injection, LN and spleen cells were isolated,
counted, and stained as described in Materials and
Methods. Mean percentages (a and
b) and numbers (c and
d) ± SEM of splenic CD4 and CD8 T cells expressing
V
), SEA/anti-OX40 (
). Mice were injected
with 0.25 mg of anti-CD40 or rat IgG on day -2 and with 0.15 µg
of SEA (a dose smaller than in Fig. 1
