By
From the Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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Evidence indicates that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We
report here that cross-linking of CTLA-4 induces transforming growth factor (TGF-
) production by murine CD4+ T cells. CD4+ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-
after antibody cross-linking of CTLA-4, indicating that induction of TGF-
by
CTLA-4 signaling represents a ubiquitous feature of murine CD4+ T cells. Stimulation of the
CD3-T cell antigen receptor complex does not independently induce TGF-
, but is required
for optimal CTLA-4-mediated TGF-
production. The consequences of cross-linking of
CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon
(Th1) and IL-4 (Th2).
Moreover, addition of anti-TGF-
partially reverses this T cell suppression. When CTLA-4
was cross-linked in T cell populations from TGF-
1 gene-deleted (TGF-
1
/
) mice, the T
cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data
demonstrate that engagement of CTLA-4 leads to CD4+ T cell production of TGF-
, which,
in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-
, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4+ T cell activation.
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Introduction |
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Recent evidence indicates that CTL-associated antigen
4 (CTLA-4),1 a counterreceptor in addition to CD28
for the B7 family of costimulatory molecules, is a negative
regulator of T cell activation (1), although this remains
controversial (5). In vitro, antibody cross-linking of
CTLA-4 has been shown to inhibit T cell proliferation and
IL-2 production triggered by anti-CD3 and anti-CD28 antibodies, whereas soluble intact or Fab fragments of anti-
CTLA-4 antibody enhance the proliferative response (2, 6-
8). In vivo, blockade of CTLA-4 with soluble intact or Fab
antibody fragments greatly enhances the T cell response to
peptide antigen (9) or superantigen Staphylococcus enterotoxin B (10). Furthermore, in vivo administration of antibodies to CTLA-4 promotes tumor rejection (11) and
exacerbates disease in experimental autoimmune encephalomyelitis (12). Direct evidence of a critical regulatory role
for CTLA-4 comes from CTLA-4-deficient (/
) mice,
which manifest a severe T cell proliferative disorder with
multiorgan lymphocytic infiltration and tissue destruction. The CTLA-4
/
animals die by 3-4 wk of age (13, 14).
These results indicate that CTLA-4 is predominantly a
negative regulator of T cell responses. However, the basis
for the inhibitory effects mediated by CTLA-4 signaling
has not yet been well elucidated. One report demonstrated that CTLA-4 mediates antigen-specific apoptosis of human
T cells (15), whereas others indicate that the primary effect
of CTLA-4 ligation is not the induction of apoptosis, at
least in murine CD4+ T cells (2, 8). In addition, T cells in
CTLA-4
/
mice exhibit normal levels of apoptosis (16).
TGF- elicits diverse cellular responses depending on
cell type, state of differentiation, culture conditions, and
presence of other cytokines (17). TGF-
is a potent inhibitor of the T cell-mediated immune response both in
vitro (20, 21) and in vivo (22), although it can also exert upregulatory effects on T cell proliferative responses
under some conditions (26). Interestingly, mice deficient in the TGF-
1 gene also develop massive lymphoproliferative disorders that lead to organ failure, and die at
the age of 2-3 wk (29), similar to the pathogenesis in
CTLA-4
/
mice. TGF-
is synthesized by different types
of cells, including T cells (20, 25, 32), although the molecular basis and pathway(s) involved in T cell production of
TGF-
are less clear.
Based on the overlapping manifestations in mice deficient in either TGF-1 or CTLA-4 and their shared immunosuppressive actions, we sought in this study to examine whether TGF-
is involved in CTLA-4 signaling of T
cells. Our results demonstrate that the cross-linking of
CTLA-4 is an effective inducer of TGF-
production by
murine CD4+ T cells in vitro. TGF-
produced by CD4+
T cells, in turn, partially suppresses the T cell proliferative response. These findings establish a previously unrecognized link between CTLA-4-mediated T cell suppression
and TGF-
.
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Materials and Methods |
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Animals.
C57BL/6 (B6) mice were purchased from The National Cancer Institute, National Institutes of Health (Bethesda, MD). TGF-Antibodies and Reagents.
Hamster unconjugated or PE-conjugated anti-murine antibodies to CTLA-4 (clone UC10-4F10-11), CD3 (clone 145-2C11), CD28 (clone 37.51), and purified IgG isotypic control antibody (clone G235-2356) were purchased from PharMingen (San Diego, CA). Rat anti-murine FITC-CD4 (clone CT-CD4), FITC-CD8 (clone CT-CD8a), and the respective isotypic control mAbs were purchased from Caltag Laboratories, Inc. (San Francisco, CA). Mouse anti-TGF-Preparation of CD4+ T Cells.
Spleens of B6 and asymptomatic TGF-CD4+ T Cell Line and Clones.
A CD4+ T cell line (CW-SW-1) and CD4+ T cell clones 2F9 (Th0), 1A11 (Th0), 1G3 (Th2), and 1C5 (Th1) specific for chicken OVA peptide 323-339 were generated from BALB/c mice adoptively transferred with CD4+ T cells of OVA TCR transgenic mice (33; a gift from Dr. D. Loh, Washington University School of Medicine, St. Louis, MO) in a tolerance model by intrathymic injection of antigen (34). All the clones express the specific clonotypic TCR (V
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Induction of TGF-.
T Cell Proliferation and Cytokine Induction.
The T cell activation assays were performed as described (7) with modifications where indicated. CD4+ T cells were incubated with the indicated antibodies in 200 µl of complete DMEM in round-bottomed 96-well plates. Anti-CD3 was added at a final concentration of 2 µg/ ml, anti-CD28 at 5 µg/ml, and anti-CTLA-4 or control hamster IgG at 20 µg/ml. For cross-linking, polyclonal goat anti-hamster antibody was added at a final concentration of 20 µg/ml. For the T cell proliferation assay, cells were then cultured at 37°C and 5% CO2 for 72 h and pulsed with 1 µCi of [3H]thymidine (NEN Research Products, Boston, MA) for the last 8-16 h. Incorporated radioactivity was counted in a beta counter (Wallac, Gaithersburg, MD). For cytokine induction, cell-free supernatants were collected at 48 h for the determination of IL-2, IFN-Cytokine ELISAs.
Quantitative ELISAs for IL-2, IFN-Flow Cytometry Analysis.
1-5 × 105 cells were resuspended in PBS without Ca2+ and Mg2+ (BioWhittaker, Inc.) containing 1% BSA (Irvine Scientific, Santa Ana, CA) and 0.1% sodium azide (Sigma Chemical Co.) (PBS-Az). For the staining of surface antigens, cells were incubated with FITC-conjugated anti-murine CD4 and PE-conjugated anti-CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® (Becton Dickinson). 104 events were routinely collected and analyzed using Lysis II software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Both the percentage of positive cells and the mean fluorescence intensity of the cells were determined.Analysis of Cell Viability.
For cell viability assays (8), T cells were cultured exactly as for the proliferation assay. After 24 and 72 h of culture, cell viability was examined by the addition of one-tenth volume of 0.4% trypan blue, and cell numbers incorporating or excluding trypan blue were determined using a hemocytometer. SDs of the duplicate wells were typically <10%.Staining DNA of Apoptotic Cells with 7-AAD.
Staining of apoptotic cells with 7-amino-actinomycin D (7-AAD) purchased from Calbiochem Corp. (La Jolla, CA) was performed by the method described by Schmid et al. (35). In brief, cells were first stained for surface antigen with FITC-conjugated anti-CD4 mAb on ice for 30 min. After washing, cells were incubated with 20 µg/ml 7-AAD in PBS-Az for 20 min at 4°C protected from light. Cells were then analyzed by FACScan® without further washing using log scale for FL-3 acquisition to assess 7-AAD staining. A minimum of 104 events was collected on each sample. Multiparameter data analysis was performed with Lysis II software. CD4+ T cells were gated, and 7-AAD staining on FL-3 versus forward scatter channels was displayed. 7-AAD staining is divided into 7-AADnegative, 7-AADdim, and 7-AADbright, representing live, early apoptotic, and later apoptotic or dead cells, respectively.Statistical Analysis.
Statistical significance of data was analyzed by Student's t test. ![]() |
Results and Discussion |
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Stimulation of freshly
purified CD4+ T cells from naive B6 mice in the presence of
anti-CD28 enhances cell growth, and as reported (2, 6),
cross-linking of CTLA-4 together with the CD3-TCR complex and CD28 effectively inhibits T cell proliferation
(Fig. 1 A) and IL-2 production (Fig. 1 B). Moreover, we
also show CTLA-4-mediated reduction of IFN- (Th1)
and IL-4 (Th2) (Fig. 1, C and D). Similar results were obtained using CD4+ Th1 and Th2 clones for these studies,
suggesting a common pathway of suppression (Chen, W.,
and S.M. Wahl, unpublished results). Consistent with previous reports (2, 8), the inhibition of CD4+ T cell activation by engagement of CTLA-4 could not be attributed to
enhanced cell death. No significant increase in apoptosis of CD4+ T cells cross-linked by anti-CTLA-4 mAb was detected either by staining of apoptotic cells with DNA dye
7-ADD for flow cytometry (Fig. 2 A) or by quantifying viable and nonviable cells (Fig. 2 B) at 24-72 h after cell culture. These data implicate alternative mechanisms of suppressed cell growth.
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To
determine whether TGF- was involved in the suppression,
CD4+ T cells purified from spleens of naive B6 mice were
added to wells containing immobilized anti-CD3 mAb.
More than 90% of these CD4+ T cells are CD44
, CD69
,
CD45RBhi, and CD62L+ and failed to proliferate to immobilized anti-CD3 mAb alone (data not shown), consistent
with their naive/resting status. Although anti-CD3 mAb did
not increase detectable TGF-
in the supernatant compared
with that of cells cultured without anti-CD3 (Fig. 3 A),
more than a fourfold increase in TGF-
levels above the
medium control was induced by the addition of anti-murine
CTLA-4 mAb followed by cross-linking with goat anti-
hamster antibody. This effect was CTLA-4 specific and not
due to the nonspecific binding of immunoglobulin, because
the control hamster antibody had no such effect. Antibody
cross-linking of CD3 and CD28, a regimen for optimal activation of naive T cells (36), failed to significantly upregulate
TGF-
production. However, when anti-CTLA-4 mAb
was added to CD4+ T cells stimulated with immobilized
anti-CD3 together with anti-CD28, a marked (10-fold) increase in TGF-
occurred (Fig. 3 A). Cross-linking CTLA-4
without CD3-TCR complex failed to increase TGF-
production (data not shown).
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Two potential mechanisms may account for the TGF-
production induced by anti-CTLA-4 antibody: stimulation
of CTLA-4 signaling through antibody cross-linking or, alternatively, blockade of CTLA-4 signaling by the antibody.
Since we used a goat anti-hamster antibody for cross-linking, the possibility of CTLA-4 signal transduction was favored. To further examine this hypothesis, purified CD4+
T cells were stimulated with the same regimen as for Fig. 3
A, but without the goat anti-hamster antibody. No TGF-
was detected in the supernatants of these cell cultures (data
not shown), consistent with a requirement for CTLA-4
cross-linking to signal TGF-
production. CD3, which
does not directly induce TGF-
production, may nonetheless facilitate CTLA-4-induced TGF-
secretion. Although CD3 alone only minimally enhanced CTLA-4 (6;
Fig. 3 B), CD3 plus CD28 stimulation markedly increased
the expression of CTLA-4 on CD4+ T cells (Fig. 3 B),
thereby promoting CTLA-4 signaling and TGF-
induction.
Since it was evident that engagement of CTLA-4
triggered TGF- production in naive/resting T cells, we
next analyzed whether cross-linking of CTLA-4 provided a
sufficient signal to induce TGF-
production by effector/
memory T cells (37) represented by murine CD4+ T cell
clones generated from a tolerance model by intrathymic injection of antigen (Chen, W.J., manuscript in preparation). Clone 1G3, which expresses clonotypic TCR specific to
chicken OVA peptide 323-339 as determined by mAb KJ
1-26 (33, 34), produces antigen-specific IL-4 but no IFN-
,
and is characterized as a Th2 clone (Table 1). Peak surface
expression of CTLA-4 on clone 1G3 was observed at 48 h
after restimulation with anti-CD3 antibody or OVA in
the presence of syngeneic irradiated BALB/c splenic APCs
(data not shown). Similar to naive CD4+ T cells, no detectable TGF-
was found in the supernatants of the clone
cells, whether stimulated with immobilized anti-CD3 alone
or with anti-CD28 antibodies. Surprisingly, cross-linking of CTLA-4 together with CD3 resulted in the highest
level of TGF-
production (Fig. 4). In contrast to naive/
resting CD4+ T cells, cross-linking of CTLA-4 with CD3
plus CD28 did not further augment TGF-
levels compared with supernatants of the clone cells stimulated with
anti-CTLA-4 and anti-CD3 (Fig. 4 A). Similar results were obtained when the OVA-specific CD4+ Th1 clone
1C5 and Th0 clone 2F9 were cultured with the same regimen of antibody stimulation (Fig. 4 A). These results indicate that the induction of TGF-
by CTLA-4 signaling is
probably a ubiquitous feature of murine CD4+ T cells. A
clear dose dependence of CTLA-4 antibody was found in
the induction of TGF-
by CD4+ T cells. With increasing
concentrations of anti-CTLA-4 antibody, TGF-
production was proportionally enhanced (Fig. 4 B).
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The discrepancy in the requirement for CD28 for
CTLA-4-induced TGF- production between naive and
effector (clone) CD4+ T cells is of interest. It is possible
that the requirement for costimulator CD28 during restimulation of CD4+ effector T cells is greatly diminished (38)
compared with naive CD4+ T cells (37). Optimal activation and, therefore, CTLA-4 expression of CD4+ naive T
cells require the coengagement of CD3-TCR complex
and CD28 (2, 6; Fig. 3 B), whereas signaling of effector
cells via CD3-TCR with the respective mAb appears independently sufficient to activate CD4+ T cell clones (37)
and CTLA-4 expression. Costimulation of CD28 failed to
further increase CTLA-4 expression (data not shown).
In the next
series of experiments, we examined the effects of anti-
CTLA-4 cross-linking on TGF- production by T cells
stimulated with antigen in the presence of syngeneic APCs.
The CD4+ T cell line CW-SM-1, specific to OVA peptide
323-339, was restimulated with OVA and irradiated
splenic APCs in the presence of anti-CTLA-4 or isotypic
hamster IgG control antibodies. Under these conditions, CTLA-4 can be cross-linked by anti-CTLA-4 via its Fc
fragment binding to Fc receptors on APCs (2, 6), which
markedly enhanced antigen-specific TGF-
production
(Fig. 5). It was noted that low but detectable TGF-
was
also found in the supernatants of cells stimulated with antigen and APCs in the absence of anti-CTLA-4 mAb. It was
unlikely that this TGF-
induced by OVA stimulation was
derived from the irradiated APCs in the culture, since no TGF-
was detected in the cultures of the T cell line and
APCs without the antigen (Fig. 5). Rather, the TGF-
induced by antigen and APCs can more likely be attributed
to the engagement of CTLA-4 by its natural B7 family
ligands on APCs. However, whether and how these B7-1
(CD80), B7-2 (CD86), and/or B7x (3, 5, 15, 36) molecules contribute to TGF-
production by engaging CTLA-4 signaling remain to be determined. Consistent with our
previous data, addition of anti-CD28 mAb in this system
failed to upregulate OVA-specific TGF-
production (data
not shown). Importantly, these results demonstrate that
cross-linking/engagement of CTLA-4 is able to induce
TGF-
production by CD4+ T cells stimulated with peptide antigen presented by APCs, as well as with specific
antibody.
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A critical question that arises is whether the suppression of T cell activation caused by CTLA-4 engagement can, in fact, be attributed to the emergence of TGF-. Since TGF-
is a potent inhibitor for T cell-mediated responses (19, 21, 39) and cross-linking CTLA-4 induces substantial levels of TGF-
(Fig. 3 A), it is reasonable to envision that TGF-
may play
a part in suppression of CD4+ T cell activation initiated by
CTLA-4 cross-linking. Consistent with the kinetics of
TGF-
production in CD4+ T cells (20), we have observed that suppression of CD4+ T cell activation due to
cross-linking of CTLA-4 is delayed. Similar kinetics of suppression have been reported using the same CTLA-4 cross-linking antibody (2), although a different mAb induces an
earlier response (24 h; reference 8). Whether this difference
represents recognition of different epitopes by the antibodies is unclear, but TGF-
appears to contribute to the suppression. First, anti-TGF-
neutralizing mAb included in
the CD4+ T cell proliferation assay, but not the isotypic
control IgG, partially restored CD4+ T cell proliferation
in a dose-dependent fashion. The suppression caused by
CTLA-4 engagement (12,287 cpm) was partially restored (nearly threefold) by the inclusion of anti-TGF-
antibody
(32,587 cpm), although it remained lower than the control
proliferative response (anti-CD3 + anti-CD28 + hamster
IgG: 118,229 cpm; Fig. 6). Second, we compared CD4+
T cells from TGF-
1
/
mice with their wild-type
TGF-
1+/+ littermates for activation with anti-CD3 and
anti-CD28 mAbs together with CTLA-4 cross-linking. As
shown (Fig. 1 A), CTLA-4 engagement profoundly inhibited T cell proliferation (90-96% suppression) in the wild-type T cells, in contrast to T cells from TGF-
1
/
mice
(Fig. 7). Although TGF-
1
/
T cells are less proliferative
in vitro than wild-type T cells (31) using optimal conditions of anti-CD3 and anti-CD28 antibodies for the wild-type and making comparisons difficult, we adjusted the
anti-CD3 and anti-CD28 concentrations to increase proliferation of the TGF-
1
/
CD4+ T cells (Fig. 7). Similar to wild-type CD4+ T cells, anti-CD3 and anti-CD28
mAbs induced significant expression of CTLA-4 on the
TGF-
1
/
T cells after 48 h of culture (>50% of CD4+
T cells become positive; Fig. 7 A). However, CTLA-4
cross-linking only suppressed the TGF-
1
/
T cell proliferation by 30-40% (Fig. 7 B), supporting the hypothesis that TGF-
1 contributes to CTLA-4-mediated suppression of T cell activation. Since engagement of CTLA-4 in
TGF-
1
/
CD4+ T cells still results in some degree of
suppression of T cell proliferation, other inhibitory molecules may also be associated with CTLA-4 signaling. Collectively, these data indicate that stimulation of CTLA-4
inhibits CD4+ T cell activation, at least in part, by inducing TGF-
production, which then downregulates their
immune responsiveness.
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Our results that cross-linking of anti-CTLA-4 mAb induces TGF- production, which then reduces T cell activation, offer an explanation for the mechanisms whereby
the CTLA-4 molecule functions as a negative regulator of
T cell responses in vitro (2, 6). This finding also has implications in our understanding of the function of CTLA-4
in T cell immune responses in vivo.
Despite the apparent unrelatedness of the two molecules,
emerging evidence has begun to link TGF- and CTLA-4.
Genes for both CTLA-4 (2, 3) and TGF-
(for reviews,
see references 39 and 40) are highly conserved among
mouse, rat, and human species. Engagement of CTLA-4
results in profound inhibition of T cell activation triggered
by TCR stimulation, including suppression of T cell cycle
(2, 8), decreased IL-2 production, and reduction of both
Th1 and Th2 cytokines (Fig. 1). TGF-
has a similar profile of activities (19, 20, 41). Neither engagement of
CTLA-4 (2, 8, 16) nor inclusion of TGF-
(37) induces
apoptosis in murine CD4+ T cell cultures. Recombinant
IL-2 is able to reverse the inhibition induced either by
CTLA-4 cross-linking (2) or by exogenous TGF-
(20;
Chen, W., and S.M. Wahl, manuscript in preparation). Most importantly, disruption of TGF-
1 (29, 30) or
CTLA-4 (13, 14) genes in mice leads to massive inflammation, multiorgan lymphocyte infiltration, and spontaneous
T cell activation. Both genotypes are associated with death
at the age of 2-4 wk. Now our data further strengthen this
link by demonstrating that CTLA-4 acts as a negative regulator by inducing TGF-
and possibly other inhibitory
molecules in T cells.
In vivo, the pathways whereby anti-CTLA-4 antibody
functions remain to be determined, although either blockade or stimulation of CTLA-4 by the injected antibodies
may occur. Blockade of CTLA-4 signaling by the antibody may lead to a loss of TGF- production by T cells.
Since TGF-
contributes to immune tolerance, suppression of autoimmune diseases in animal models (23-25, 42-
44), and inhibition of antitumor immunity of T cells infiltrating into tumors (45), loss of TGF-
production and
removal of this potent inhibitor of T cells could release a
"brake" on T cell activation. Consequently, augmentation
of the T cell antitumor immune response (11) and exacerbation of autoimmune diseases such as experimental autoimmune encephalomyelitis due to increased IL-2 and IFN-
production may occur (3, 12). On the other hand, if anti-
CTLA-4 antibody given in vivo cross-links CTLA-4 on T
cells via its Fc fragment on FcR+ cells, T cells might commence production of TGF-
to effect immune suppression, tolerance (49), and inhibition of tumor growth (50-
52). Thus, based on our in vitro data, it is now possible to reevaluate the in vivo impact of CTLA-4 antibodies
on TGF-
production and their cooperative roles in mediating antitumor responses, immunoregulation, and tolerance.
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Footnotes |
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Address correspondence to Sharon M. Wahl, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bldg. 30, Rm. 332, Bethesda, MD 20892. Phone: 301-496-4178; Fax: 301-402-1064; E-mail: smwahl{at}yoda.nidr.nih.gov
Received for publication 8 July 1998 and in revised form 14 September 1998.
We thank Drs. Nancy McCartney-Francis and Gillian Ashcroft for helpful discussions and critical review of the manuscript, Dr. S. Khoury (Brigham and Women's Hospital, Boston, MA) for support, and George McGrady for maintaining the mice. We also thank Dr. D. Loh for providing the TCR transgenic mice.
Abbreviations used in this paper 7-AAD, 7-amino-actinomycin D; B6, C57BL/6; CTLA-4, CTL-associated antigen 4.
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