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Department of Microbiology, Oregon State University, Corvallis, OR 97331
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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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To mount an optimal T cell response against Ag, it is thought that APC deliver both signals to T cells. These two signals activate T cells and drive T cell clonal expansion. As a result, many T cells are generated, and, of those, many become effector T cells 11 . Normally, non-APC do not bear the B7 molecules and thus cannot deliver signal two 12 . Under such conditions, based on in vitro data with T cell clones, delivery of signal one without signal two does not activate the T cell fully, but instead directs it to a nonresponsive state known as anergy 3, 13 . Perhaps surprisingly, little is known about T cell activation in vivo. In part this is due to the complexity of APC/T cell interactions and the vast number of signals each cell can deliver to each other. These include not only "costimulatory" signals but also the variety of cytokines that are secreted.
Although in an optimal T cell response clonal expansion is important for fighting pathogens, just as important is the ability to down-regulate the T cell response after the pathogen is cleared. T cell populations must decline in number or else autoimmunity and immunopathology can occur 14, 15 . Some cells must survive, however, if T cell memory is to develop. Thus, a complex balance must be maintained between sustained immunity and tolerance in the immune system.
Efficient monitoring of the T cell response, both expansion and deletion, has been performed with the use of staphylococcal enterotoxin A (SEA)3. SEA is a SAg that binds MHC class II and selectively engages all TCRs that contain the Vß3 chain 16, 17, 18, 19, 20 . Injection of B10.Br mice with SEA promotes the clonal expansion of CD4 and CD8 T cell populations that bear Vß3; however, these cells very soon afterward decrease in number to below normal uninjected levels 21, 22 .
In this study, we set out to use the SEA model to investigate a very important molecule whose role in the immune system is only beginning to be made clear: CD40 23 . CD40 is a member of the TNF receptor family 24 . It is expressed on all APC such as B cells 24 , macrophages 25 , and dendritic cells 26 and has also been reported on T cells 27, 28 . CD40 ligand (CD40L, CD154, gp39) is a member of the TNF family as well 29, 30 . It is expressed mainly on CD4 T cells 31 but is also found on CD8 T cells 31, 32, 33 as well as other cells such as eosinophils and NK cells 34, 35 .
Ligation of CD40 has been shown to yield many effects on APC. Several groups have shown that CD40 ligation can enhance the costimulatory abilities of APC 36, 37, 38 . In fact, other studies have shown that lack of CD40 ligation can actually promote T cell tolerance as a result of poor B7 expression 39, 40 . Many groups have investigated the role this tolerance mechanism plays in autoimmunity and transplant rejection. For example, autoimmune conditions such as experimental allergic encephalomyelitis 41, 42 , collagen-induced arthritis and insulin-dependent diabetes mellitus 43, 44 are augmented by CD40 activation. Other studies have found that stimulation of CD40 plays a role in graft transplant rejection 45, 46, 47 .
Effects of CD40 activation on T cell activation have also been reported. For example, IL-12-dependent Th1 differentiation has been shown to be modulated by CD40 activation 38, 48 . Most recently, CD40 ligation has been shown to circumvent the requirement for T helper cells in the priming of CTL 49, 50, 51 . CD8 T cell responses are thus also affected by CD40 ligation. Observed effects include improved antitumor-killing abilities 52, 53 and memory CTL responses 53, 54 . Little has been reported on death susceptibility of T cells after CD40 ligation; however, recent reports have shown that CD40 activation of B cells, monocytes, and dendritic cells can block apoptosis within these populations 55, 56 .
To examine the effects of CD40 stimulation on T cell clonal expansion and deletion upon response to an Ag, an anti-CD40 agonistic mAb was used in conjunction with SEA injection. We found that activation of CD40 not only enhanced CD4 and CD8 T cell clonal expansion but also delayed, but did not prevent, their subsequent deletion. Thus, CD40 enhances an immune response in vivo by increasing the number of effector T cells and delaying their subsequent death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female B10.Br/SgSnJ and B10.A/Cr mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and the National Cancer Institute (Frederick, MD), respectively, and maintained in our animal facility under specific pathogen-free conditions. In all experiments, mice between the ages of 6 and 12 wk were used.
Reagents, experimental protocols, and Abs
SEA was purchased from Toxin Technology (Madison, WI) and administered to mice as i.p. injections of 0.15 or 0.30 µg. The anti-CD40-producing hybridoma FGK45.5 was a kind gift from Dr. Ton Rolink (Basel Institute, Switzerland) 57 . The Ab was purified from hybridoma supernatants over protein G columns (Pharmacia, Piscataway, NJ). As a control, rat IgG (Sigma, St. Louis, MO) was injected in doses equal to the anti-CD40.
In experiments designed to block interactions between CD28 and B7-1/B7-2, CTLA4IG 58 or an isotype-matched control chimeric Ab, L6, was injected i.p. at 0.50 mg/injection. These were both kind gifts from Dr. Peter Linsley (Bristol Myers-Squibb, Seattle, WA). Alternatively, Abs directed against B7-1 (16-10A1; 59 or B7-2 (GL1; 6 were used at doses of 1 mg/injection. These hybridomas were obtained from ATCC (Manassas, VA), and the resulting Abs were purified separately from hybridoma supernatants over protein G columns (Pharmacia).
Anti-TCR Vß3 (KJ25-607.7; 59 and anti-IEk (14.4.4; 61 were purified separately from hybridoma supernatants over protein G columns (Pharmacia). These Abs were FITC conjugated by us. FITC-conjugated annexin V, FITC-conjugated anti-Vß14, PE-conjugated anti-CD4, PE-conjugated streptavidin, and biotinylated anti-TCR Vß3 were all purchased from PharMingen (San Diego, CA). PE-conjugated anti-B7-1, PE-conjugated anti-B7-2, PE-conjugated anti-macrophage Ab (F4/80), and PE-conjugated anti-CD45R (B220) were all purchased from Caltag (Burlingame, CA). Red 613-conjugated anti-CD4 and Red 613-conjugated anti-CD8 were purchased from Life Technologies (Grand Island, NY).
Injection schedule
Injection of SEA was at time 0 h, and all other injections were done in relation to SEA. Injections before SEA injection were designated as negative days, while injections after SEA were designated as positive days. The anti-CD40 Ab was injected on day -2 before SEA unless otherwise stated. Rat IgG was always given to a control group at the same time as anti-CD40 was given to an experimental group. The molecules L6, CTLA4IG, anti-B7-1, and anti-B7-2 were all given 2 h before SEA. All injections were i.p.
Cell processing and flow cytometry
Spleens were removed and teased through nylon mesh (Falcon, Becton Dickinson, Franklin Lakes, NJ) and subjected to ammonium chloride to lyse red blood cells. Peripheral LNs (inguinal, axillary, and bronchial) were teased into single cell suspensions and washed with balanced salt solution (BSS). T cells from spleen or LN populations were purified on nylon wool columns as described previously 60 . Briefly, 3-cc syringes were filled with 0.12 to 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 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 Fc
receptor mAb (24.G2; 61 and 10 µg/ml human 
-globulin (Sigma)
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
second staining step was necessary, the incubation and wash procedures
were repeated. Flow cytometry was conducted on an EPICS XL flow
cytometer (Coulter Electronics, Miami, FL). Greater than 5000 viable
cells were analyzed with WinList software (Verity Software House,
Topsham, ME).
Histochemistry
The mesenteric LN and spleen from each mouse were fixed in PBS 4% paraformaldehyde. They were transferred into OmniSette tissue cassettes (Fisher Scientific, Pittsburgh, PA) and washed with distilled water for 2–3 h and then soaked in 70% ethanol overnight. The tissues were embedded in paraffin after a 1-h wash in 85% ethanol, three 1-h washes in 95% ethanol, three 1-h washes in 100% ethanol, and three 1-h washes in xylene. Paraffin-embedded tissues were sectioned and baked onto Superfrost/Plus microscope slides (Fisher Scientific) overnight. Tissues were stained using the Apoptosis Detection System, Fluorescein from Promega (Madison, WI), which uses the TdT-mediated dUTP Nick-End Labeling (TUNEL) assay 62 . Briefly, tissues were deparaffinized with organic solvents and permeabilized with proteinase K. The tissues were then incubated with TdT enzyme and a nucleotide mixture containing FITC-labeled dUTP. This was done in a humidified chamber for 1 h at 37°C. Tissues were then washed, counterstained with propidium iodide, and then sealed under a cover slip. Confocal images were captured at x20 magnification using a Leica TCS 4D confocal microscope (Heidelberg, Germany) and combined using Adobe Photoshop software (Mountainview, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial studies sought to examine the effects CD40 activation would have on T cell populations in the presence of stimulating Ag. To examine this issue, mice were treated with SEA. SEA is a SAg that stimulates T cells bearing TCR Vß3 chains 16 ; therefore, SAg-stimulated T cells can be directly analyzed after SEA treatment. Mice were injected either with SEA alone or with SEA and anti-CD40. As a control for the SEA/anti-CD40 group, a third group was injected with SEA and rat IgG. A final group was left uninjected (normal) as a negative control. Fourteen days after SEA injection, the peripheral (inguinal, axillary, and bronchial) LN and the mesenteric (mucosal) LN, as well as the spleen, were removed, and the T cells were isolated by nylon wool fractionation separately from each tissue. The T cells were stained for CD4 Vß3 and CD8 Vß3 expression and analyzed by flow cytometry.
The results show that the presence of Ag alone causes significant
deletion of both CD4 and CD8 T cells bearing Vß3 in each tissue
examined 14 days after SEA (Fig. 1
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Percentages of each T cell population were two- to fourfold lower in
SEA-injected mice than in uninjected controls. In contrast, injection
of anti-CD40 in SEA-treated mice potently inhibited
Ag-induced T cell deletion in every tissue examined. CD4 Vß3 and CD8
Vß3 percentages in these mice were generally as high as, if not
higher than, those in the uninjected control, and in all
cases higher than those in the mice injected with SEA alone.
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). The results showed the same amount
of T cell deletion that was observed with SEA alone, strongly
suggesting that CD40 activation blocks SAg-induced T cell deletion.
Percentages can be misleading due to migratory effects and bystander T
cell death, so it was important to examine the absolute number of T
cells in the various lymphoid organs. Data shown in Table I confirm that deletion was inhibited,
since CD4 Vß3 numbers are elevated in the anti-CD40- and
SEA-treated mice compared with SEA alone. An additional control
experiment was performed where anti-CD40 alone was injected. The
results showed no differences in T cell percentages when compared with
an uninjected control (data not shown). Furthermore, staining of the T
cells from each tissue for TCR Vß14 chains, a subset of T cells that
does not respond to SEA, showed no significant difference in their
percentages when compared with the uninjected control (data not shown).
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To standardize future experiments with anti-CD40, the optimal
conditions for anti-CD40 injection were determined. Experiments
were set up in which B10.Br mice were injected with 1 mg of
anti-CD40 at different times in relation to SEA. Anti-CD40
injections were performed at 5 days before SEA injection (day -5) and
up to 1 day after (day +1). Eight days after SEA injection, T cells
from the LN (Fig. 2) and spleen (not
shown) were counted, stained for CD4 Vß3 and CD8 Vß3, and analyzed
by flow cytometry.
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, A and C). Most
injections of anti-CD40 were effective at preventing the deletion,
regardless of the day injected relative to SEA. Deletion was most
efficiently prevented when anti-CD40 was injected before SEA. For
example, when anti-CD40 was injected on day +1, the percentage of
CD4 Vß3 T cells was 3.8 ± 0.9%; when anti-CD40 was
injected on days -2, -3, and -4, however, this rose to 11.6 ±
2.4%, 9.0 ± 2.5%, and 9.1 ± 3.1%, respectively (Fig. 2A). The percentages of CD8 Vß3 T cells were above normal
in every case, except when anti-CD40 was injected 5 days before SEA
(Fig. 2C). The CD4 Vß3 percentages were near or above
normal, except when anti-CD40 was given on day -5 and day +1. All
SEA/anti-CD40 injections consistently led to greater Vß3 T cell
percentages than those observed with SEA alone. Based on percentages,
day -2 was the best day for anti-CD40 injection. On this day, CD4
Vß3 percentages rose to 11.6 ± 2.4%, almost six times the
level observed in SEA alone-treated mice (2.1 ± 0.5%) (Fig. 2A). CD8 Vß3 percentages rose from a 1.5 ± 0.6%
population found in SEA injected mice to 9.4 ± 1.6% (Fig. 2C).
The total numbers of CD4 Vß3 (Fig. 2B) and CD8 Vß3 (Fig. 2D) T cells were calculated. Days -2, -3, and -4 had the
highest total numbers of Vß3 T cells after SEA/anti-CD40
treatment. Day -2 was still the best, providing counts of 9.4 x
105 ± 4.0 and 4.9 x 105 ± 1.9 CD4 and
CD8 T cells bearing Vß3, respectively (Fig. 2, B and
D). Based on these results, day -2 was chosen as the
standard day of injection of anti-CD40, since it gave the most
consistently high percentages and numbers of both CD4 Vß3 and CD8
Vß3 T cells.
Once the timing of anti-CD40 injection was determined, the dose of anti-CD40 was titrated. One mg of anti-CD40 was injected into each mouse for the timing experiments. We tested whether lower doses would still be effective at preventing Ag-specific T cell deletion. Experiments were set up in B10.Br mice, which were injected with anti-CD40 two days before receiving SEA. Anti-CD40 was injected at 1 mg, 0.5 mg, 0.25 mg, or 0.125 mg. An uninjected control mouse and a mouse receiving SEA only were also included. Seven days after SEA was injected, the LN and spleen T cells were isolated, counted, and stained for CD4, CD8, and Vß3. T cell analysis was done by flow cytometry.
LN-dosing experiments are shown in Fig. 3, and spleen data are similar, but not
shown. The resulting percentages show the deletion of CD4 Vß3 (Fig. 3A) and CD8 Vß3 (Fig. 3C) T cells in the LN
upon injection of SEA alone. All doses of anti-CD40 were effective
at preventing deletion, as shown by the percentages (Fig. 3, A and C) and numbers (Fig. 3, B and
D). The highest dose tested (1 mg) was the most effective at
generating high T cell numbers, but even an eightfold lower dose of
anti-CD40 provided some T cell rescue. Both percentages and numbers
show that the CD8 Vß3 T cells were rescued more effectively by lower
doses of anti-CD40 than were CD4 Vß3 T cells. A dose of 0.25 mg
yielded equivalent numbers of CD8 Vß3 T cells as the 1-mg dose
(5 x 105 ± 0.6) (Fig. 3D). In contrast,
CD4 Vß3 T cell deletion was less inhibited by lower doses of
anti-CD40 (Fig. 3B) but was still very effective. Based
on these data, 0.25 mg of anti-CD40 was chosen as the standard dose
in future experiments.
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Since anti-CD40 inhibited the deletion of Ag-specific T cells
exposed to SEA (
Figs. 1–3), we next investigated whether anti-CD40
affected the expansion and long-term deletion of Vß3 T cells by
conducting a detailed time course. In this experiment, mice were
injected with 0.25 mg of anti-CD40 or, as a control, 0.25 mg of rat
IgG. Two days later, SEA was injected into each group. On days 2, 5, 7,
12, and 21 after SEA injection, LN and spleens from both groups of mice
were obtained. T cells were purified from these tissues, counted,
stained for CD4 Vß3 and CD8 Vß3, and analyzed by flow cytometry.
The control mice injected with SEA and rat IgG (squares) show some CD4
Vß3 T cell expansion (day two), and significant deletion by day five
(Fig. 4). Examination of numbers shows a
small degree of expansion in the LN (Fig. 4C) but a greater
than twofold increase in the spleen (Fig. 4D). The degree of
expansion is quite variable depending on dose and batch to batch
variation of SEA (our unpublished observations). After clonal
expansion, T cell populations deleted quickly. By day 5, both
percentages (Fig. 4, A and B) and numbers (Fig. 4, C and D) fell below normal and stayed there
until day 21. The slight rise in T cell numbers at the end of the time
course is most likely due to repopulation of deleted T cells from the
thymus.
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A) and a large increase in the
spleen (Fig. 4B) to about three times starting levels on day
2. By day 5, both tissues were showing T cell expansion to about three
times control levels. T cell percentages stayed above the rat IgG
control levels throughout the time course but did decline to control
levels as day 21 approached. Based on numbers, the LN show about a
fourfold expansion of the CD4 Vß3 T cells by day 5, only to decline
to near control levels by day 12 (Fig. 4C). Spleen numbers
were not much greater than controls on day 2, but T cell deletion was
greatly inhibited until day 21 (Fig. 4D).
The CD8 Vß3 time course results closely resembled the CD4 Vß3 data
(Fig. 5). The percentages and numbers of
CD8 Vß3 LN or spleen T cells from SEA/rat IgG-injected mice slightly
increased on day 2, followed by deletion starting after day 2.
SEA/anti-CD40-injected mice again showed increased expansion and
delayed deletion. Both percentages (Fig. 5, A and
B) and numbers (Fig. 5, C and D)
showed greater than fourfold increases over the levels seen in SEA/rat
IgG-injected mice on days 5 and 7 but came back down to control levels
by day 21. It is important to note that, as a negative control, Vß14
T cells were also examined during this time course. There was little
variation in Vß14 T cell populations when compared with uninjected
mice (data not shown).
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Stimulation of CD40 delays T cell death
In each of the previous experiments, it was shown that injection of anti-CD40 inhibited the Vß3 T cell deletion characteristic of SEA stimulation. These data raise the possibility that CD40 activation delays the death of the SEA-stimulated T cells.
To determine whether T cell death was inhibited by CD40 activation, mice were treated as follows: injected with SEA alone, anti-CD40 alone, SEA and anti-CD40, or left as uninjected controls. On days 3 and 9 after SEA injection, the mesenteric LN and spleen from each mouse were fixed in PBS containing 4% paraformaldehyde and stained for apoptotic cells as described in Materials and Methods. Confocal microscopy was used to generate images of each stained tissue.
Fig. 6, A-D, shows
LN results 3 days after SEA injection, while Fig. 6, E-H, shows the results from day 9. On day 3, SEA
alone led to significant apoptosis (Fig. 6B), some of which
was still observed 9 days later (Fig. 6F). Injection of
anti-CD40 alone yielded low levels of apoptosis on both days 3 and
9 (Fig. 6, C and G, respectively). Injection of
SEA and anti-CD40 led to low levels of death on day 3 (Fig. 6D) but enhanced apoptosis by day 9 (Fig. 6H).
These data show that injection of anti-CD40 and SEA does delay cell
death, but they do not indicate whether the dead cells are
Ag-stimulated T cells or not.
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were
crushed, and the T cells were isolated and stained for CD4, Vß3,
Vß14, and extracellular phosphatidylserine (PS). PS is displayed
extracellularly on dying cells and can be detected by staining with its
natural ligand, annexin V, and analyzed by flow cytometry (Fig. 7) 63 .
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A); thus, significant death was occurring at this early
time point in comparison with uninjected animals (8.5 ± 0.5%).
Mice injected with anti-CD40 alone, or with SEA and anti-CD40,
remained about 2.5-fold lower (6.8 ± 2.7% and 7.1 ± 2.8%,
respectively). On day 9, death had more than doubled in SEA-injected
mice to 40.2 ± 3.9%; however, it should be kept in mind that
these mice contained far fewer Vß3 T cells on day 9 than on day 3
(see time course data in Figs. 4 and 5). Mice injected with
anti-CD40 alone showed only a slight increase (Fig. 7A).
The percentage of death in mice injected with SEA and anti-CD40
after 9 days (39.1 ± 3.3%) was nearly equal to that in mice
injected with SEA alone, rising about sixfold from day 3. The day 9
mice treated with SEA and anti-CD40 also contained a greater number
of Vß3 T cells than the SEA alone group (Figs. 4 and 5). As a
control, CD4 Vß14 T cells were also examined for extracellular PS
expression (Fig. 7B), and no major changes above normal
percentages were observed in any of the groups. These data suggest that SAg-stimulated T cells begin to die early after injection with SEA and will continue along that path for days afterward. Activation of CD40 delayed the death of those SAg-specific T cells.
B7-1 and B7-2 play different roles in CD4 and CD8 T cell stimulation in the presence of SEA and CD40 activation
Since CD40 activation delayed SAg-specific T cell death, we sought to test the mechanism by which CD40 was acting. One possibility was that CD40 activation altered APCs. To examine this hypothesis, three separate experiments were performed in which one mouse was injected with anti-CD40 and another mouse was given rat IgG. Two days later, the LNs and spleens were isolated, and the cells were stained to identify B cells and macrophages. Expression of MHC class II, B7-1, and B7-2 on these cells was examined by flow cytometry.
Mean channel fluorescence (MCF) of MHC class II expression increased
approximately fourfold on both macrophages and B cells obtained from
the LN and spleen of mice injected with anti-CD40 compared with rat
IgG (data not shown). Both B cells and macrophages also up-regulated
B7-1 and B7-2 in the spleen (Fig. 8) as
well as the LN (data not shown) of mice treated with anti-CD40
compared with mice treated with rat IgG. Specifically, splenic MHC
class II+ B220+ B cell MCF of B7-1 (16.7) and
B7-2 (18.0) increased by more than 2.5-fold over control cells (6.6 and
5.5, respectively). MCF MHC class II+ F4/80+
macrophage expression of B7-1 (17.6) and B7-2 (22.0) in
anti-CD40-treated mice increased by more than fivefold over control
levels (3.5 and 3.9, respectively). These data are similar to those
previously reported 64 . Thus, one possibility was that CD40
activation was assisting T cell expansion by increasing expression of
B7-1 and/or B7-2 on APC.
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and previous reports 64, 65 , we next tested the
hypothesis that CD40 mediated T cell activation in vivo through B7. We
first sought to inhibit the CD28-B7 interaction using CTLA4IG. One
group of mice received SEA only; one received SEA, anti-CD40 and
CTLA4IG; and another group received SEA, anti-CD40, and the control
chimeric Ab L6. Five days after SEA injection, T cells from the LN and
spleen of each mouse were isolated, counted, and stained for CD4 Vß3
and CD8 Vß3. The cells were analyzed by flow cytometry.
Mice injected with SEA alone showed the expected decrease in CD4 Vß3
and CD8 Vß3 expansion in the LN and spleen (Table II). Injection of SEA, anti-CD40, and
L6 led to significant expansion over normal levels in both CD4 and CD8
T cell populations in the LN and spleen, similar to that shown in Figs. 4 and 5. When CTLA4IG was injected with SEA and anti-CD40, however,
CD4 Vß3 percentages were lowered in both tissues, although never to
the level of mice injected with SEA alone. CD4 T cell populations in
the spleen and LN decreased twofold, (22.1 ± 0.6% to 11.1
± 0.6%, and 16.0 ± 0.9% to 7.9 ± 1.2%, respectively).
CD8 Vß3 percentages differed from the CD4 percentages in that they
were only slightly inhibited by CTLA4IG injection. Spleen percentages
decreased from 17.8 ± 0.9% to 15.1 ± 1.4%, while the LN
percentages dropped from 13.7 ± 0.6% to 11.7 ± 1.1%
(Table II).
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). The splenic CD4 Vß3 T cell
numbers in SEA/anti-CD40/L6-injected mice (30.6 x
105 ± 2.5) dropped to uninjected levels (8.9 x
105 ± 0.4) when CTLA4IG was given. CD8 Vß3 T cell
numbers did show more of a decline than the percentages did, especially
in the spleen, falling from 22.3 x 105 ± 1.6 in
SEA/anti-CD40/L6-treated mice to 14.9 x 105 ±
1.7 in mice treated with CTLA4IG. The numbers of T cells, however, was
still almost fourfold greater than uninjected or SEA-injected (4.2
x 105 ± 0.3) mice (Table III).
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Both percentages and numbers of CD4 Vß3 T cells showed the expected
deletion of SEA-activated T cells and the expected enhanced expansion
of those Vß3 T cells upon CD40 stimulation, both in the LN (Fig. 9 A and C) and
spleen (Fig. 9, B and D). Injection of
anti-B7-1 led to a modest drop in both percentages and numbers,
while injection of anti-B7-2 led to a slightly greater decrease.
When both anti-B7-1 and anti-B7-2 were injected, CD4 Vß3 T
cell populations dropped below uninjected control levels, close to
SEA-injected levels. For example, in the LN-uninjected mice there were
2.7 x 105 ± 0.2 CD4 Vß3 T cells, while in mice
treated with both Abs there were 2.0 x 105 ± 0.3 T
cells.
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). The decrease
and increase of CD8 percentages and numbers upon SEA injection and
SEA/anti-CD40 injection, respectively, were still noted in both the
LN (Fig. 10, A and C) and spleen (Fig. 10, B and D). Injection of anti-B7-1 did little
to inhibit anti-CD40-mediated T cell expansion. Anti-B7-2
consistently dropped the percentages and numbers, suggesting that it
was the dominant molecule for CD8 Vß3 T cell clonal expansion. These
data show that B7-1 costimulation in the context of SAg and CD40
activation is less important for CD8 T cells than it is for CD4 T
cells. Additionally, the increased drop in CD8 T cell expansion
observed when blocking B7-1 and B7-2 may be due to the low levels of
CD4 T cell help available.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial timing experiments provided many important clues as to the
mechanism by which CD40 activation enhances a T cell response. The
timing data show that stimulation of CD40 before SAg exposure creates
the conditions for an optimal T cell response to foreign SAg (Fig. 2).
Conversely, CD40 activation does not enhance SAg-induced T cell clonal
expansion when it occurs after SAg exposure. One explanation for the
latter result is that the T cells have already interacted with SAg/MHC
and, thus, APC are unable to costimulate activated T cells outside the
context of SAg in vivo. Therefore, it is likely that stimulating CD40
before Ag injection helps create a better costimulatory environment on
APC, as evidenced by the enhanced expression of B7-1 and B7-2 on B
cells and macrophages (Fig. 8) as well as increased MHC class II
expression (data not shown). These primed APC can enhance T cell
stimulation. We have, however, recently found B cells to be unessential
for the increased T cell expansion in this model, since a similar
response occurs in B cell knockout mice (our unpublished observations).
Without stimulation of CD40, the SEA-activated T cells clonally expand
to peak levels within 2 days and delete to below starting levels after
about day 5 (Figs. 4 and 5). Activation through CD40 in the presence of
SAg, however, enhances the clonal expansion observed on day 2 and
delays the time it takes for the T cell populations to delete to
control levels to about 21 days. Additionally, we show that low doses
of anti-CD40 worked very well at enhancing clonal expansion and
delaying SAg-specific T cell deletion (Fig. 3). Collectively, the
results in this study show that acute activation of CD40 is sufficient
to enhance T cell clonal expansion in peripheral and mucosal lymphoid
tissue.
One of the most intriguing effects of CD40 activation was its ability to delay SAg-specific T cell deletion. We hypothesized that the observed deletion was due to death. Deletion during negative selection in the thymus has recently been shown to be due to apoptosis 69 . Likewise, in the periphery, reports have shown that peptides presented by MHC promote apoptosis of TCR transgenic T cells 70 .
Tissue sections from mice in which apoptotic cells were detected showed
that there was indeed a greater level of apoptosis observed in
SEA/anti-CD40-treated mice 9 days after SEA injection (Fig. 6).
Without anti-CD40, SEA induced death by 72 h in vivo. Because
cell death occurred, it was determined whether the dying cells were
indeed SEA-specific T cells. We confirmed this by examining Vß3 T
cell populations for extracellular PS expression by staining with
annexin V (Fig. 7). Not only did CD40 activation enhance T cell clonal
expansion, but, additionally, the stimulated T cells were viable for
longer periods of time. Ligation of CD40, however, does not keep the T
cells alive indefinitely; it only delays death. B cells have also been
reported to receive a "life" signal when CD40 is activated 55 .
Collectively, these data suggest that CD40 activation increases the
lifespan of effector T cells by delaying their death.
Once the effects of CD40 activation on T cell responses to SAg had been examined, a role for costimulation in this response was addressed. CD28 is a costimulatory molecule found on T cells that binds B7-1 and B7-2 molecules on APC. CD28 knockout mice show several T cell deficiencies, including poor T cell proliferation and IL-2 production 71, 72, 73 . Thus, the importance of CD28 ligation in the SEA/anti-CD40 model was studied.
Using CTLA4IG to block the ligation of CD28 by B7 molecules, we found a decrease in SEA-specific T cell expansion in vivo (Tables II and III). This, taken along with the time course data, suggests that CD28 ligation is important for T cell expansion, but not for long term survival, since the cells still go on to die even with CD28 ligation. Because CD8 T cell responses were less inhibited by CTLA4IG, it became interesting to examine the roles of B7-1 and B7-2 separately in this model to begin uncovering the costimulatory dependence of each T cell subpopulation.
B7-1 and B7-2 both bind CD28 and CTLA4 6, 7, 10 . Since B7-1 and B7-2 behave in a similar manner, extensive work has been conducted to determine what different functions B7-1 and B7-2 may have. To date, results have been contradictory. It has been held that ligation of B7-1 can skew CD4 T cell development in the Th1 direction, while ligation of B7-2 promotes Th2 development 74, 75 . However, others have found no differential effects in T cell proliferation or cytokine production depending on the B7 molecule ligated 76, 77 . Data showing contrasting effects between B7-1 and B7-2 stimulation in CD8 T cell development are also contradictory 76, 78, 79 ; however, Xu et al. have found less of a dependence on B7-1 in CTL activation 80 . Currently, B7-2 appears to be a more important molecule in that it is often found expressed earlier in an immune response 81, 82 and is expressed on murine memory CD4 T cells 83 . These latter data suggest that memory cells may be able to costimulate other T cells, resulting in quicker responses to Ag. Although our data do not resolve these issues, they do provide in vivo evidence that CD8 T cells depend more on B7-2 for clonal expansion than B7-1.
Several groups have recently reported that CD40 activation can actually bypass the helper T cell requirement in CTL activation 49, 50, 51 . Their in vivo and in vitro data have found that CTL killing activity was normally dependent on CD4 T cell function, yet this requirement for T cell help could be circumvented by CD40 ligation. Interestingly, we show that CD40 activation enhances CD8 T cell expansion and delays their subsequent death in vivo. Thus, it may be that more CTLs are active when CD40 is activated, which will thus generate a greater CTL activity due to the higher numbers of CTLs. Additionally, our data suggest that one way CD40 activation may bypass the T helper cell requirement for CTL function is by keeping the activated CTL alive for longer periods of time so that a greater number of cellular targets can be destroyed. Ridge et al. further found that signaling via B7-1 or B7-2 was necessary, but not sufficient for CTL activity 49 . We observed a minor requirement for CD28 ligation in driving CD8 T cell clonal expansion. It may be that the CD28-B7 interaction is less important for SAg-driven CD8 T cell clonal expansion than it is for actual killing activity.
An important question in immunology today is centered around
identifying factors that are important in overriding tolerogenic
responses. This is important not only as a basic immunological
question, but also as a method to achieve better vaccination protocols
and prevention of autoimmunity. The use of SAg to study central and
peripheral tolerance is widely accepted. Injection of SAg into mice
leads to clonal expansion of the reactive T cells followed by profound
deletion 16 . Here, it is shown that SEA does induce death and that
this death can be delayed by coactivating CD40-bearing cells.
Ultimately, the frequency of SEA-specific cells was not increased by
day 21, regardless of whether anti-CD40 was injected with SEA
(Figs. 4 and 5). The significance of these data is underscored when one
considers that the CD40-bearing APC were indeed well activated since
they bore large quantities of B7 and MHC class II, well over that of
mock-activated cells (Fig. 8 and data not shown). Thus, these data
suggest that APC activation is not enough to permanently override a
tolerogenic response. The mechanistic data in this report show
unequivocally that CD40 activation drives T cell activation through
ligation of CD28. Therefore, the data show that CD28 ligation is not
sufficient to force a long term T cell response in this model. Hence,
tolerance induction is the default pathway, and overriding this type of
a response cannot be accomplished through Ag stimulation in conjunction
with CD28 ligation.
For the most part these data are dissimilar to much of the data that
have been garnered in vitro. For years it has been suggested that the
difference between long term immunity and tolerance is ligation of CD28
84 . Many laboratories have shown that Ag stimulation in the absence
of CD28 ligation led to anergy, whereas TCR and CD28 stimulation broke
this tolerogenic response 85, 86 . These data are almost exclusively
from in vitro studies and have been very difficult to test in vivo.
Recent studies have shown that CD28 ligation occurred when SAg alone
was injected into mice 66 . Those studies raised several issues that
were not addressed until now. First, it was unclear whether B7-1 and/or
B7-2 was involved, and the response of CD8 T cells was not addressed in
those studies. Moreover, it was unclear whether the amount of CD28
ligation was sufficient. This is an important point since it is known
that APC up-regulate B7 after activation. Thus, activated APC may be
able to deliver a qualitatively and quantitatively different signal to
cognate T cells. For example, it is possible that ligation of CD28 in
response to SAg alone was minimal in the absence of APC activation. In
the present study we tested this idea more directly by activating APC
with a potent agonist mAb specific for CD40. Under these circumstances
it is clear that APC activation was profound, and, based on the data
shown in Figs. 4, 9 and 10, CD28 was ligated significantly over that
when SEA alone was injected. Perhaps surprisingly, this treatment was
still unable to break tolerance even though clonal expansion was far
greater when CD40 was activated. Therefore, CD28 ligation, be it
minimal or presumably maximal, with TCR stimulation in vivo does not
promote long-term T cell survival but only enhances expansion and
delays subsequent death in this model.
These data raise the question of what can block T cell deletion. As
shown previously, coinjection of bacterial LPS is capable of inhibiting
Ag-induced deletion 87 . This response was shown to occur
independently of CD28 ligation but was profoundly dependent on TNF-
production 66, 87 . In contrast, the CD40 mAb response shown here
drives CD4 responses almost entirely through CD28 ligation. One
possibility is that CD40 activation does not induce as much TNF- as
LPS. Also it is possible that a different pattern of cytokines is
produced when comparing the two types of responses. Ultimately, it may
be that the combination of TNF- and CD40 activation may synergize to
yield an optimal response. For example, CD40 may drive the clonal
expansion phase, and TNF- may prevent death by delivering a survival
signal to the activated cells. These ideas are currently being tested
by our laboratory. Nevertheless, these data suggest that vaccine
development should rely not only on CD28 ligation or APC activation
through CD40, but also on treatments that prime for long term T cell
survival.
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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, 220 Nash Hall, Department of Microbiology, Oregon State University, Corvallis, OR 97331. E-mail address:
3 Abbreviations used in this paper: SEA, staphylococcal enterotoxin A; SAg, superantigen; LN, lymph node; PE, phycoerythrin; PS, phosphatidylserine; MCF, Mean channel fluorescence.
Received for publication August 31, 1998. Accepted for publication November 5, 1998.
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) or
rat IgG (
) 2 days before receiving 0.15 µg of SEA. T cells were
purified after SEA injection on days 2, 5, 7, 12, and 21 from the LN
(A and C) and spleen (B
and D). T cells were stained for CD4 Vß3 and analyzed
by flow cytometry. Each point represents the mean percentages
(A and B) and numbers (C
and D) ± SEM from three mice from one representative
experiment of two separate experiments.
