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Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was due to their impaired IL-12 production. From these
results, we conclude that TGF-ß2 treatment impairs the
ability of macrophages to produce IL-12 and to express CD40. As a
consequence, TGF-ß2-treated PEC fail to promote
development of pT cells toward the Th1 phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß, a highly conserved homodimeric 25-kDa protein, is a potent immunoregulatory agent that affects T cell proliferation (15, 16, 17), activation state (18, 19), and differentiation (20, 21, 22). However, the majority of TGF-ß studies fail to distinguish whether the cytokine’s modulatory effect on T cell behavior is direct, or indirect by virtue of TGF-ß’s ability to influence APC function. The earlier studies on ACAID highlighted this issue since only APC, not T cells, were exposed directly to TGF-ß. We have, therefore, initiated experiments to examine in vitro the extent to which TGF-ß modifies APC function. To that end, we have reported recently that TGF-ß2 prevents PEC from stimulating Ag-specific T cells to secrete Th1 cytokines, although TGF-ß2-treated PEC as well as untreated PEC stimulate T cell proliferation. In part, this occurs because TGF-ß2 induces PEC to secret TGF-ß in an autocrine fashion, which modifies the functional program of the responding T cells such that Th2-type lymphokines are selectively produced (23). However, in these experiments, neutralization of TGF-ß (produced by TGF-ß2-treated PEC) failed to restore completely the ability of TGF-ß2-pretreated PEC to stimulate T cell secretion of Th1-type cytokines. These findings suggest that 1) other factors are required for proper Th1-type cytokine stimulation, and 2) their expression is impaired in PEC treated with TGF-ß2.
IL-12, a 70-kDa heterodimeric cytokine composed of linked p35 and p40
chains, is produced by monocytes/macrophages (24), and has
immunomodulatory effects on T cells and NK cells (25, 26, 27, 28). IL-12
stimulates IFN- synthesis and proliferation by T cells and NK cells
(25, 29), alone or in synergy with other factors such as IL-2 (28).
These properties and the ability of IL-12 to promote Th1 and inhibit
Th2 responses among human PBLs stimulated with allergens or bacterial
Ags (27), and OVA-specific TCR transgenic T cells to OVA (30), alerted
us to the potential importance of IL-12 in ACAID induction.
CD40, a cell surface molecule found on APCs including B cells,
dendritic cells, and macrophages, is a receptor for CD40 ligand (CD40L)
expressed on activated T cells. When ligation occurs, a signal is
delivered to APC that triggers IL-12 production (31, 32, 33, 34, 35). In this
study, we examined whether treatment of PEC with TGF-ß2
inhibits their ability to produce IL-12 and to express costimulatory
molecules, and whether changes are involved in the altered
Ag-presenting abilities of PEC treated with TGF-ß2. Our
results indicate that TGF-ß impairs the ability of PEC to produce
IL-12 and to express CD40. Moreover, low CD40 expression reduces the
ability of cocultured Ag-activated T cells to induce enhancement of
IL-12 production by PEC. This accounts for the inability of
TGF-ß-treated PEC to induce pT cells to produce IFN- upon Ag
stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal female BALB/c mice at 6 to 8 wk of age were purchased from Taconic Farms (Germantown, NY) and used as a source of PEC. DO11.10 TCR transgenic mice were maintained in our colony (original parents were a kind gift of Dr. Dennis Loh, Washington University, St. Louis, MO) and used as the source of T cells in the present study. DO11.10 mice express the DO11.10 TCR that is specific for the peptide fragment of OVA, 323–339, in the context of I-Ad (30, 36).
Serum-free medium
Serum-free medium was used for cell culture. It was composed of RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Biowhitaker, Walksville, MD), and 1 x 10-5 M 2-ME (Sigma Chemical Co., St. Louis, MO), and supplemented with 0.1% BSA (Sigma Chemical Co.), ITS + culture supplement (1 µg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 µg/ml Fe(NO3)3) (Collaborative Biomedical Products, Bedford, MA).
DO11.10 T cell purification
Spleens and lymph nodes were removed from DO11.10 TCR transgenic mice and pressed through nylon mesh to produce a single cell suspension. RBC were lysed with Tris-NH4CL. Then they were washed three times with RPMI 1640, and purified by T cell enrichment column (R&D Systems, Minneapolis, MN). The recovered cells were >95% Thy-1+ cells.
Preparation of PEC pretreated with TGF-ß2
PEC were obtained from normal BALB/c mice that received 2 ml of
thioglycolate (Sigma Chemical Co.) i.p. 3 days earlier. PEC were
washed, resuspended, placed in either 96-well flat-bottom plate (1
x 105/well) or 24-well culture plate (1 x
106/well), and treated overnight with or without 5 ng/ml of
porcine TGF-ß2 (R&D Systems) in serum-free medium at
37°C in an atmosphere of 5% CO2. After overnight
culture, plates were washed three times with culture medium to remove
TGF-ß2 and nonadherent cells. Approximately 60% of the
cells added initially remained adherent in the wells and were used in
all subsequent experiments. More than 90% of these adherent cells were
F4/80+ (shown in Fig. 8
).
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To assay for content of IL-4, IL-10, and IFN-, DO11.10 T
cells (2.5 x 104/well) were added into the
96-well plates containing pretreated PEC, and cultured with or without
varying concentrations of native OVA (Sigma Chemical Co.) in serum-free
medium. In some experiments, IL-12, anti-IL-12 Ab (C17.8),
anti-IL-4 Ab (11B11), and anti-CD40L Ab (MR1) (PharMingen, San
Diego, CA) were also added to these cultures. Cells were cultured for
48 h at 37°C in an atmosphere of 5% CO2; then
supernatants were collected and analyzed by quantitative capture ELISA,
according to the manufacturer’s instructions (PharMingen). For IL-12
assay, TGF-ß2-treated or untreated PEC (1 x
106/well) in 24-well culture plates were cultured alone, or
TGF-ß2-treated or untreated PEC (1 x
105/well) in 96-well micro plates were cultured with
DO11.10 T cells (2.5 x 104/well) and a range of
concentrations of native OVA. In some of the latter experiments,
anti-CD40L Ab (MR1) (PharMingen) was also added to these cultures.
Twenty-four hours later, supernatants were harvested and assayed for
the presence of IL-12 by quantitative capture ELISA. Rat mAbs to mouse
cytokine IL-4 (BVD4-1D11), IL-10 (JES5-2A5), IFN- (R4-6A2), or IL-12
(C15.6) were purchased from PharMingen and used as coating Abs.
Biotinylated rat mAbs to mouse cytokines IL-4 (BVD6-24G2), IL-10
(SXC-1), IFN- (XMG1.2), or IL-12 (C17.8) (PharMingen) were used as
detecting Abs.
Quantitative RT-PCR for IL-4, IL-10, IFN-, and IL-12p40 mRNA
Total cellular RNA from 1 x 106 PEC or T cells purified by T cell enrichment was extracted by treatment with acid guanidium thiocyanate-phenol-chloroform using the RNA stat-60 kit (Tel-Test, Friendswood, TX). Purified total RNA (approximately 100 ng) was reverse transcribed in 20 µl of reverse-transcription mixture (100 mM KCl, 20 mM Tris-HCl, 25 mM MgCl2, 1 mM dNTP, 100 µM hexanucleotides, and 4 U per reaction of avian myeloblastosis virus reverse transcriptase (AMV-RT)) and incubated for 1 h at 42°C and 5 min at 95°C. Quantitative PCR amplification was then conducted: briefly, 2 µl of cDNA was added to 50 µl of amplification mixture (50 mM KCl, 10 mM Tris-HCl (pH 9 at 25°C), 2 mM MgCl2, 0.2 mM dNTP, 0.01% gelatin, and 0.1% Triton X-100) containing 0.25 µM sense and antisense primers and 1.25 U/reaction Taq polymerase (Perkin-Elmer, Foster City, CA), and the mixture was overlaid with mineral oil (Sigma chemical Co.). The thermal profile was initially at 94°C for 3 min, followed by 42 cycles at 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min in a thermal cycler (model PTC-100; M-J Research, Watertown, MA). At sequential cycle numbers, 5 µl of the reaction mixture was sampled through oil and transferred onto avidin-coated microtiter plates containing 95 µl of Tris-EDTA buffer for quantitation of the amplified products by a liquid hybridization-ELISA assay, as previously described (37). Briefly, we used 0.2 pmol of digoxigenin-labeled probe per well, conjugated anti-digoxigenin alkaline phosphatase Ab, and paranitrophenyl substrate at 1 mg/ml in 1 M diethanolamine buffer. The absorbance was measured at 405 nm using an ELISA plate reader, and the color change was determined. The results were expressed as the ratio of lymphokine cDNA to GAPDH cDNA.
Oligonucleotides
All probes and primers were synthesized and biotinylated by
Bioserve Biotechnologies (Laurel, MD) and used without further
purification. The amplicon-specific probes were digoxigenin labeled
using the digoxigenin oligonucleotide 3'-end labeling kit, according to
manufacturer’s instructions (Boehringer Mannheim Corp., Indianapolis,
IN). Primers and probe sequences (designed by Dr. Alard with the
exception of the IL-12 primers) are listed in Table I
and were synthesized as shown (5' to
3').
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TGF-ß2-treated or untreated adherent cells alone, or cultured with DO11.10 T cells and OVA (100 µg/ml) for 24 or 72 h, were collected and incubated with CD16/CD32 Fc block (PharMingen, San Diego, CA) in 2% normal mouse serum-PBS for 10 min on ice. The cells were then stained with 1/50 dilution of FITC-conjugated anti-F4/80 mAb, and 1/20 dilution of PE-conjugated anti-B7-1 mAb, anti-B7-2 mAb, or anti-CD40 mAb (PharMingen, San Diego, CA) for 30 min on ice. FITC-conjugated nonspecific rat IgG2b and PE-conjugated nonspecific rat IgG2a were used as isotype controls. They were then washed twice with 2% normal mouse serum-PBS. Labeled cells were analyzed on an EPICS XL flow cytometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly prepared PEC were cultured overnight with or without 5
ng/ml of TGF-ß2, and washed extensively to remove
TGF-ß2 and nonadherent cells. With this protocol, more
than 90% of adherent cells were F4/80-positive macrophages (Fig. 8
).
TGF-ß2-treated or untreated PEC were cocultured with
DO11.10 T cells in the presence of a range of Ag concentrations. After
48 h, the cell supernatants were analyzed for the presence of IL-4
and IFN-. Results from a representative experiment are displayed in
Figure 1. Untreated PEC failed to
stimulate DO11.10 T cells to secrete IL-4, but stimulated large amounts
of IFN- secretion in an Ag dose-dependent manner. In contrast,
TGF-ß2-treated PEC failed to stimulate IFN- secretion,
but stimulated DO11.10 T cells to secrete IL-4 at 100 µg/ml of OVA.
Neither untreated PEC nor TGF-ß2-treated PEC were able to
induce DO11.10 T cells to secrete IL-10 (data not shown).
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mRNA
expression by quantitative RT-PCR using a liquid hybridization assay
(37). Results from a representative experiment are displayed in Figure 3. DO11.10 T cells stimulated with untreated PEC expressed high levels
of IFN- mRNA, but low levels of IL-4 mRNA. In contrast, DO11.10 T
cells stimulated with TGF-ß2-treated PEC expressed high
levels of IL-4 mRNA, but no IFN- mRNA (Figs. 2, A and B).
There was no difference in IL-10 mRNA expression between DO11.10 T
cells stimulated with untreated PEC and those stimulated with
TGF-ß2-treated PEC (data not shown).
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production by DO11.10 T cells stimulated with
TGF-ß2-treated APC in the presence of neutralizing
anti-IL-4 Ab
Since IL-4 has been shown to inhibit production of IFN- by pT
cells, it is possible that IL-4 produced by DO11.10 T cells stimulated
with TGF-ß2-treated PEC inhibits IFN- production
by DO11.10 T cells. In this regard, we examined whether addition of
anti-IL-4 Ab to the cell culture will restore IFN- production by
DO11.10 T cells when stimulated by TGF-ß2-treated PEC. We
cultured DO11.10 T cells with TGF-ß2-treated PEC in the
presence of neutralizing anti-IL-4 Ab, and measured IFN- content
in the culture. Neutralization of the IL-4 secreted by DO11.10 T cells
in response to stimulation with TGF-ß2-treated PEC (Fig. 3A) did not permit
IFN- secretion by DO11.10 T cells (Fig. 3B). These
data suggest that factors other than IL-4 may be involved in the
impairment of IFN- production.
Influence of TGF-ß2 on IL-12 production by APC
IL-12, which is produced by certain APC, has been shown to promote
the differentiation of naive T cells into the Th1 phenotype, and to
inhibit the differentiation of IL-4-producing Th2 cells (27, 38).
Therefore, we hypothesized that the altered Ag-presenting abilities of
TGF-ß2-treated PEC demonstrated above might result
from an inhibition of their IL-12 production. To investigate whether
TGF-ß2 impairs IL-12 production by PEC, quantitative
RT-PCR using a liquid hybridization assay was performed. Both the p35
and the p40 chains of IL-12 are necessary for a functional IL-12
molecule. The p40 chain is inducible upon stimulation, while the p35
chain is constitutively produced and only slightly inducible by certain
stimuli (39, 40). Therefore, we chose to examine IL-12 p40 mRNA
expression in untreated and TGF-ß2-pretreated PEC. Figure 4 displays the results obtained from a
representative experiment. The results indicate that the level of IL-12
p40 mRNA in TGF-ß2-pretreated PEC was approximately
threefold lower than that observed in untreated PEC.
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A). As the results
shown in Figure 5B indicate, when DO11.10 T cells and OVA
were added to wells containing TGF-ß2-treated or
untreated PEC, the amount of IL-12 detected in the supernatants was
much higher. However, in the presence of DO11.10 T cells, IL-12
secretion by TGF-ß2-treated PEC remained significantly
lower than that of untreated PEC. We conclude from these data that
TGF-ß2 treatment significantly impairs IL-12 production
by PEC, as well as altering the enhancement of IL-12 secretion mediated
by interaction with T cells and Ag.
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production by DO11.10 T cells stimulated with
TGF-ß2-treated APC in the presence of exogenous
IL-12
If the ability of TGF-ß2-treated PEC to promote
DO11.10 T cell secretion of IL-4 rather than IFN- was due to
impaired IL-12 production, exogenous IL-12 should restore these altered
Ag-presenting abilities. To test this possibility, DO11.10 T cells were
cultured with OVA-pulsed, TGF-ß2-pretreated PEC in the
presence or absence of a range of concentrations of exogenous IL-12.
Results from a representative experiment are shown in Figure 5. On the
one hand, as little as 100 pg/ml of exogenous IL-12 was able to restore
completely the ability of TGF-ß2-pretreated PEC to induce
DO11.10 T cells to secrete IFN- (Fig. 6A). On the other hand,
100 pg/ml of exogenous IL-12 completely abolished the ability of
TGF-ß2-pretreated PEC to stimulate IL-4 secretion by
DO11.10 T cells (Fig. 6B). These results demonstrate
that exogenous IL-12 is able to restore the Ag-presenting abilities of
PEC pretreated with TGF-ß2, thus enabling them to
stimulate DO11.10 T cells to produce IFN- rather than IL-4. In
addition, the amounts of exogenous IL-12 required to restore the
Ag-presenting ability of TGF-ß2-treated PEC are similar
to the decreased levels of IL-12 observed following
TGF-ß2 pretreatment (Fig. 5).
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production by DO11.10 T cells stimulated with
untreated APC in the presence of anti-IL-12 Abs
If, as the previous results suggest, a deficit in IL-12 is
critical to the altered Ag-presenting functions of
TGF-ß2-treated PEC, then a similar effect should be
achieved by adding anti-IL-12 Abs to cultures containing untreated
PEC. To test this premise, DO11.10 T cells were cultured with untreated
PEC in the presence of neutralizing anti-IL-12 Ab, and the amount
of IL-4 and IFN- in the supernatants was measured. The results of
representative experiments are displayed in Figure 7. In the presence of neutralizing
anti-IL-12 Ab, untreated PEC as well as
TGF-ß2-treated PEC failed to stimulate DO11.10 T cells to
secrete IFN- (Fig. 7A). However, unlike
TGF-ß2-treated PEC, untreated PEC could not stimulate
IL-4 secretion by DO11.10 T cells, when the culture contained
neutralizing anti-IL-12 Ab (Fig. 7B). Altogether
these data suggest that the deficit in IL-12 observed in
TGF-ß2-treated PEC is responsible for the failed
induction of IFN- secretion by DO11.10 T cells. However, this
deficit cannot explain the ability of TGF-ß2-treated PEC
to induce T cells to secrete IL-4, suggesting that other factors are
involved. We have shown in a previous publication that TGF-ß itself
produced by TGF-ß2-treated PEC was responsible for the
induction of IL-4-secreting cells (23).
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To this point, our findings suggest that
TGF-ß2 treatment impaired IL-12 production by PEC,
especially when Ag-activated T cells were present in the cultures. We
wondered whether APC-T cell interactions were being disrupted by
TGF-ß. Costimulatory signals arising from receptor/ligand
interactions such as B7/CD28 and CD40/CD40L are known to participate in
T cell activation. We first examined B7-1, B7-2, and CD40 expression on
the surface of TGF-ß2-treated and untreated PEC cultured
in the absence of DO11.10 T cells and OVA. Results of representative
experiments are displayed in Figure 8.
B7-1 expression was not detected by either untreated or
TGF-ß2-treated PEC, whereas B7-2 was highly expressed by
both untreated and TGF-ß2-treated PEC. However, there was
no difference in B7-2 expression between untreated PEC and
TGF-ß2-treated PEC. CD40 was expressed at low, albeit
detectable levels on cultured PEC, and its expression was consistently
lower in TGF-ß2-treated PEC than untreated PEC.
Next, we examined B7-1, B7-2, and CD40 expression by
TGF-ß2-treated and untreated PEC cultured for 24 and
72 h in presence of DO11.10 T cells and OVA. As shown in Figure 9, expression of CD40 by the analyzed
cell population (F4/80+) of untreated PEC was increased
substantially 24 h after culture with DO11.10 T cells (74.8%), in
comparison with PEC alone (21.3%; Fig. 8). CD40 expression on
TGF-ß2-treated PEC cultured with T cells plus OVA for
24 h was more modestly up-regulated (46.4%; Fig. 9). However, the
expression of B7-2 on both TGF-ß2-treated and untreated
PEC was similar 24 h after culture with DO11.10 T cells. Cells
examined by flow cytometry after 72 h of culture showed similar
results, although B7-2 expression was only marginally reduced on
TGF-ß2-treated PEC (32%) as compared with untreated PEC
(46%) (data not shown). Since there appeared to be a selective, albeit
partial, impairment of CD40 expression after TGF-ß2
treatment, we considered the possibility that CD40 expression
may be related to altered Ag-presenting abilities of
TGF-ß2-treated PEC.
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production by DO11.10 T cells stimulated with
untreated and TGF-ß2-treated APC in the presence of
anti-CD40L Abs
Since the costimulatory signal provided by CD40 has been reported
to induce IL-12 production by PEC (32, 33, 34, 35), we investigated whether the
impaired ability of TGF-ß2-treated PEC to produce
IL-12 was caused by the fact that they express less CD40 than untreated
PEC. It has been shown that anti-CD40L Ab blocks ligation of CD40
with CD40L, and prevents CD40-dependent activation (41). In this
experiment, TGF-ß2-treated or untreated PEC were cultured
for 24 h with DO11.10 T cells plus OVA in the presence or absence
of anti-CD40L Ab. IL-12 content in the supernatants was then
measured. Representative results are
displayed in Figure 10A. In the presence of anti-CD40L Ab, IL-12 production
by untreated PEC cocultured with DO11.10 T cells was reduced
significantly to levels similar to those of
TGF-ß2-treated PEC. These results suggest that 1) CD40
expression on PEC is required for optimal IL-12 production in the
presence of Ag-activated T cells, and 2) reduced CD40 expression on
TGF-ß2-treated PEC impairs the enhancement of IL-12
production observed in the presence of DO11.10 T cells.
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is due to their impaired CD40 expression that results in
suboptimal IL-12 production. We measured IFN- production by DO11.10
T cells cultured with untreated PEC plus OVA in the presence of
anti-CD40L Ab. For comparison, supernatants of T cells stimulated
by OVA and TGF-ß2-treated PEC were also examined. As
shown in Figure 10B, IFN- secretion by DO11.10 T cells
cultured with untreated PEC was decreased substantially in the presence
of anti-CD40L Ab.
We conclude that TGF-ß impairs both IL-12 production and CD40
expression by PEC, and that failed CD40 expression robs cocultured T
cells of their ability to enhance PEC production of IL-12. As a
consequence, the T cells receive inappropriate signals for IFN-
production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not IL-4,
whereas T cells exposed to TGF-ß-treated, OVA-pulsed PEC secreted
Th2-type cytokines, IL-4, but not IFN-. It was found that PEC
treated with TGF-ß produced increased amounts of TGF-ß, the
majority of which was secreted in its mature form (41). However,
neutralizing anti-TGF-ß Abs abolished the ability of
TGF-ß-treated, OVA-pulsed PEC to stimulate T cells to secrete IL-4,
indicating that endogenous TGF-ß was responsible for the potential
shift of the T cell response toward a Th2-type pattern. The
enhancement of IL-4 levels may also reflect the selective expansion of
a small population of memory T cells described in unprimed DO110 mice
that may be precommitted to make IL-4 (42), which in turn could inhibit
Th1 cell differentiation. In contrast, neutralizing anti-TGF-ß
Abs failed to completely restore the IFN- production of T cells
exposed to OVA-pulsed, TGF-ß2-treated PEC, implying that
other factors besides TGF-ß are important in inhibiting the Th1
response.
Our present experiments reveal that although T cells stimulated with
OVA-pulsed, TGF-ß2-treated PEC secrete IL-4, the
production of this cytokine in the culture is not the reason that
IFN- is not produced. Addition of neutralizing anti-IL-4 Abs to
the cultures did not permit IFN--producing T cells to emerge.
Therefore, we examined the effects of TGF-ß on the acquisition of
accessory or costimulatory signals by the PEC. IL-12, a cytokine that
is critical to the generation of IFN--secreting T cells, is produced
by cultured PEC (24, 27, 28). However, PEC pretreated with
TGF-ß2 produced significantly less IL-12 than untreated
PEC. Moreover, supernatants of cultures containing OVA-pulsed,
TGF-ß2-treated PEC and DO11.10 T cells contained far less
IL-12 than companion cultures containing untreated PEC and T cells.
These data indicate that a deficit in IL-12 generation exists in
TGF-ß2-treated cultures, and suggest that this deficit
might be important in the failure of T cells to produce IFN-. In
support of this hypothesis, we observed that the addition of exogenous
IL-12 to T cell-containing cultures stimulated with
TGF-ß2-treated PEC led to the activation of
IFN--producing T cells that failed to secrete IL-4. Thus, the
failure of TGF-ß2-treated PEC to promote T cell
differentiation toward the Th1 phenotype resulted from a deficit of
IL-12 production. In addition, at a high concentration of OVA (200
µg/ml), TGF-ß2-treated PEC induced T cells to secrete
marginally more IL-4 than untreated PEC. This drop in IL-4 secretion
observed at a high dose of Ag may be due to the higher level of IL-12
secreted under similar conditions (Fig. 5).
The capacity of APC to secrete sufficient IL-12 to drive T cell
differentiation down the Th1 pathway depends upon the ability of the
APC to express CD40 as a costimulatory signal for responding T cells
(33, 34, 35). Our experimental results indicate that inadequate CD40
expression occurs in TGF-ß2-treated PEC. First, although
fresh PEC do not express any costimulatory molecules (data not shown),
PEC spontaneously up-regulated their CD40 after being cultured in
vitro for 24 h. However, this up-regulation of CD40 expression on
PEC was lower in the presence of TGF-ß2 than in the
absence of TGF-ß2. Second, the addition of T cells to
OVA-pulsed PEC markedly enhanced CD40 expression on the PEC during the
next 24-h culture interval, whereas there was no comparable
up-regulation of CD40 on similarly cultured
TGF-ß2-treated PEC. We then demonstrated that a deficit
in CD40 expression is important in the failure of
TGF-ß2-treated PEC to secrete sufficient IL-12 to
activate IFN--secreting T cells. An Ab that blocks ligation of CD40
with its ligand significantly inhibited IL-12 production by untreated,
OVA-pulsed PEC in the presence of T cells. In these cultures, the IL-12
level was similar to that of OVA-pulsed, TGF-ß2-treated
PEC. We conclude that TGF-ß2-treated PEC produce
insufficient IL-12 to promote the differentiation of DO11.10 T cells
toward the Th1 phenotype, and that this deficit is due to the inability
of TGF-ß2-treated PEC to up-regulate CD40.
Since coculture with DO11.10 T cells dramatically up-regulated CD40 expression on untreated PEC, but not on TGF-ß2-treated PEC, it is possible that the inability of TGF-ß2-treated PEC to up-regulate CD40 was due to down-regulated CD40L expression on DO11.10 T cells stimulated with TGF-ß2-treated PEC. To investigate this hypothesis, we also examined CD40L expression on DO11.10 T cells when stimulated with TGF-ß2-treated or untreated PEC for 24 h. Although about 10% of DO11.10 T cells expressed CD40L after stimulation with TGF-ß2-treated or untreated PEC, we were not able to show a significant difference in CD40L expression between DO11.10 T cells stimulated with both untreated and TGF-ß2-treated PEC (data not shown). Therefore, these data suggested that TGF-ß2 might act directly on PEC by preventing up-regulation of CD40 at their surface.
We are at a loss to explain the selective failure of PEC treated with
TGF-ß2 to express adequate amounts of CD40. Our evidence
indicates that TGF-ß2 does not cause a global inhibition
of expression of MHC class II (data not shown) and B7-2 on APC.
Cultured TGF-ß2-treated PEC express MHC class II and B7-2
at levels that are comparable with untreated PEC. Although
TGF-ß2-treated PEC expressed less B7-2 than untreated PEC
at the end of culture period (data not shown), this may not be due to a
direct effect of TGF-ß2 treatment. Since IFN-
amplifies expression of B7-2 (32, 43), this outcome may be explained by
impaired IFN- production in cultures of TGF-ß2-treated
PEC. Experiments are underway to determine whether TGF-ß2
directly and selectively suppresses CD40 gene expression in
PEC.
Some in vivo aspects of ACAID suggest that certain responding T cells
are biased toward the Th2 phenotype. First, animals with ACAID fail to
develop and display Ag-specific DH, a Th1-mediated response (1, 2, 3).
Second, the sera of mice that receive an intracameral injection of OVA,
followed 1 wk later by s.c. immunization with OVA in CFA, contain high
levels of OVA-specific IgG1 Abs (1), but lack the complement-fixing
IgG2a Abs usually associated with Th1 responses. Third, Li et al. have
provided evidence recently that the spleen cells from mice with
OVA-specific ACAID produce IL-10 (but not IFN- or IL-4) when
stimulated in vitro with OVA (44). However, conflicting evidence also
has been reported. For example, Bando et al. found that cervical lymph
nodes of BALB/c mice bearing progressively growing intraocular P815
tumors contain DBA/2-specific T cells that proliferate and secrete IL-2
when stimulated with DBA/2 alloantigens in vitro (5). However, these
cells made neither IL-4 nor IFN-, a phenotype reminiscent of the
so-called precursor Th cell. Moreover, Kosiewicz and Streilein have
reported that the Th2 phenotype is expressed by lymphoid cells of mice
with ACAID only when the animals have been immunized with Ag plus CFA
(45). Spleen cells of mice that received only an AC injection of OVA
respond to OVA stimulation in vitro by making only TGF-ß. Thus, even
though the results of our present experiments indicate that
TGF-ß2-treated PEC promote DO11.10 T cells to
differentiate toward the Th2 phenotype, we cannot be completely sure
that this accurately reflects the in vivo situation.
We have shown previously that two populations of regulatory T cells participate in vivo in ACAID: a CD8+ T cell that effects efferent suppression of DH, and a CD4+ T cell that inhibits the induction of DH. In the present in vitro experiments, the T cells we used were obtained from TCR transgenic DO11.10 mice, and 95% of the cells were CD4+, rather than CD8+. Therefore, our data potentially address the CD4+ regulatory ACAID T cell, and suggest that it may be of the Th2 type. However, our results tell us little, if anything, about the CD8+ regulatory T cells that are also important in ACAID. Our next experimental goal is to examine the effects of ACAID-inducing APC on transgenic OVA-specific CD8+ T cells that are restricted by class I MHC molecules.
Very recently, three unexpected and provocative new observations have expanded the possible immune regulatory pathways of ACAID. Niederkorn et al. have claimed that B lymphocytes are required in the induction of ACAID (46), Cone et al. have reported that ACAID fails to occur in mice that were thymectomized before AC injection of Ag (47), and Griffith et al. found that induction of ACAID to hapten-derivatized syngeneic spleen cells fails in mice that are unable to express Fas ligand (48). Clearly, much remains to be learned before the phenomenon of ACAID submits to a full understanding.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Wayne Streilein, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford, Boston, MA 02114. E-mail address:
3 Abbreviations used in this paper: AC, anterior chamber; ACAID, anterior chamber-associated immune deviation; CD40L, CD40 ligand; DH, delayed hypersensitivity; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PE, phycoerythrin; PEC, peritoneal exudate cells.
Received for publication July 10, 1997. Accepted for publication October 23, 1997.
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