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*
Laboratory of Experimental Immunology, Departments of
Pathology and
Gastroenterology, and
Laboratory of Experimental Hematology, University Hospital Gasthuisberg, Leuven, Belgium;
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Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium;
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Tanox Pharma, Amersterdam, The Netherlands; and
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Institute of Infectious Disease and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by lamina propria CD4+ cells. In
contrast, anti-CTLA-4 treatment led to deterioration of disease, to
more severe inflammation, and to enhanced production of proinflammatory
cytokines. Of note,
CD25+CD4+ cells from
CD28-/- mice similar to those from the
wild-type mice were efficient to prevent intestinal mucosal
inflammation induced by the wild-type
CD45RBhigh cells. The inhibitory functions of
these regulatory T cells were effectively blocked by anti-CTLA-4.
These data show that the B7-CD28 costimulatory pathway is required for
induction of effector T cells and for intestinal mucosal inflammation,
while the regulatory T cells function in a CD28-independent way. CTLA-4
signaling plays a key role in maintaining mucosal lymphocyte tolerance,
most likely by activating the regulatory T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive T cell activation requires two distinct signals from the APC. The first signal is triggered by the engagement of the Ag-specific TCR-CD3 complex, and the second is a costimulatory signal. T cell recognition of Ag in the absence of costimulation may lead to anergy. CD28 is the most widely studied costimulatory molecule and is constitutively expressed on the surface of T cells. The natural ligands for CD28 have been identified as B7 molecules (B7.1 or CD80 and B7.2 or CD86) and have been demonstrated to be present or to be inducible on the surface of APC (3). CD28-B7 interaction results in an enhanced T cell proliferation, cytokine production, and resistance to apoptosis (3). Blockade of CD28-B7 interaction in vitro effectively prevents effector T cell responses (3). CD28-/- T cells exhibit defects in cell survival, clonal expansion, and differentiation into effector cells in response to Ag stimulation (4, 5).
Several lines of evidence have also demonstrated that B7-CD28
interactions play an important role in some immune diseases.
Overexpression of B7 molecules has been found in the inflamed areas
from patients with multiple sclerosis (6), rheumatoid
arthritis (7), and psoriasis (8). We and
others have reported increased expression of B7.1 and B7.2 molecules in
the inflamed mucosa of IBD patients (9, 10).
Administration of anti-B7.1 prevents the development of
experimental autoimmune encephalomyelitis (EAE) in mice
(11, 12, 13). Anti-B7.2 suppresses the onset of diabetes in
nonobese diabetic mice (14) and prevents the development
of lung mucosal allergic inflammation (15, 16). In vivo
administration of B7 blocking agents reduces the severity of autoimmune
diseases (14, 17, 18) and prolongs long-term graft
survival (19, 20). In addition,
CD28-/- mice are resistant to collagen-induced
arthritis (21) and EAE induction (22) and
show a delayed rejection of cardiac allografts (23). A
second receptor for B7, the CTLA-4 molecule, is also important in
immune regulation. CTLA-4 (CD152), a homologue of CD28, is up-regulated
on the surface of activated T cells (24). It competes with
CD28 to bind B7 and functions as a counterregulatory receptor that
attenuates T cell responses by down-regulating T cell activation
(25, 26, 27), by facilitating Ag-specific apoptosis
(28), and by suppressing secretion of both Th1 and Th2
cytokines (26, 27, 29). Blockade of CTLA-4 signaling with
neutralizing Ab was found to promote expansion of Ag-specific T cells
(25), to enhance T cell IL-2 and IFN- secretion
(29), and to augment antitumor immunity (30).
Consistent with these findings, CTLA-4-/- mice
develop an autoimmune lymphoproliferative disorder accompanied by
extensive lymphocytic infiltration into multiple organs, resulting in
tissue destruction and early lethality (31, 32). It has
further been shown that administration of blocking anti-CTLA-4
exacerbates EAE (33, 34), autoimmune diabetes
(35), and allograft rejection (23).
To study the role of B7, CD28, and CTLA-4 costimulatory molecules in the control of intestinal inflammation, we used an adoptive transfer model of colitis in mice. Transfer of CD45RBhighCD4+ (naive) T cells from BALB/c mice to syngeneic SCID mice leads to the development of an IBD-like syndrome by 6–10 wk after T cell reconstitution, characterized by diarrhea, weight loss, transmural inflammation in the proximal colon, and a Th1 immune response by lamina propria CD4+ T cells (36, 37). The clinical, histopathological, and immunological features resemble those observed in human Crohn’s disease. Of particular importance, cotransfer of the reciprocal CD45RBlowCD4+ T cells or CD25+CD4+ T cells together with pathogenic CD45RBhigh cells prevents the development of colitis (36, 38), indicating that the CD45RBlow/CD25+ subsets function as regulatory T cells to control mucosal inflammatory responses. Moreover, in vivo administration of anti-CTLA-4 has been recently reported to abrogate inhibition of colitis by CD25+CD4+ cells, suggesting that CTLA-4 signaling is involved in the immune-suppressive function of these regulatory cells (38). Targeted therapy directed against T cell costimulatory molecules such as CD40 ligand has also shed some light on the control of intestinal mucosal inflammation (36, 39). In this study, SCID mice were reconstituted with syngeneic CD45RBhighCD4+ cells from the wild-type (WT) or CD28-/- mice, and they were treated with either anti-B7.1 or anti-B7.2 or anti-CTLA-4 mAb to investigate the in vivo relevance of B7-CD28/CTLA-4 interaction to the control of intestinal inflammation. In addition, we also explored the potential role of CD28 and CTLA-4 costimulatory signals in the regulatory functions of CD45RBlow and CD25+CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female WT BALB/c mice were obtained from Harlan (Zeist, The Netherlands). Female BALB/c SCID mice were bred under standard pathogen-free conditions and maintained in the certified animal facility of the University Hospital Gasthuisberg, Catholic University of Leuven (Leuven, Belgium). Female BALB/c CD28-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were all housed in a specific pathogen-free facility in microisolator cages with filtered air and free access to food and water. All mice were 6–8 wk of age when experiments were initiated.
Antibodies
Anti-mouse CD3
(500A2, hamster IgG (HIg)), biotinylated
anti-mouse CD4 (H129.19, rat IgG2a), biotinylated anti-mouse
B7.1 (16–10A1, hamster IgG), FITC-conjugated anti-mouse CD45RB
(16A, rat IgG2a), PE-conjugated anti-mouse CD4 (GK1.5, rat IgG2b),
PerCP-conjugated anti-mouse CD4 (RM4-5, rat IgG2a), FITC-conjugated
anti-mouse CD25 (7D4, rat IgM), and PE-conjugated anti-mouse
CTLA-4 (UC10-4F10-11, HIg) were purchased from BD PharMingen (San
Diego, CA). Anti-mouse CD54 (KAT-1, rat IgG2a) was obtained from
BioSource International (Nivelles, Belgium). Biotinylated
anti-mouse F4/80 (CI:A3-1, rat IgG2b) was purchased from Serotec
(Oxford, U.K.). Hamster anti-mouse B7.1 (16-10A1, IgG)
(40), rat anti-mouse B7.2 (GL1, IgG2a)
(41), and hamster anti-mouse CTLA-4 mAb (UC10-4F10-11)
(25) were purified from culture supernatants of the
hybridomas, which were obtained from the American Type Culture
Collection (Manassas, VA). HIg and rat IgG2a as controls were purchased
from BioTrend (Cologne, Germany).
Purification of T cell subsets
CD4+ T cells and
CD45RBhigh and
CD45RBlowCD4+ T cells were
isolated from BALB/c spleen using a method as previously reported
(36). Briefly, splenic CD4+ T cells
were isolated by positive selection using mouse CD4 Dynabeads (L3T4)
and mouse CD4 DETACHaBEAD (Dynal, Oslo, Norway). These procedures
resulted in 
98% CD4+ T cells as assessed on a
FACSort (Becton Dickinson, San Jose, CA). CD4+ T
cells were then stained with FITC-conjugated anti-CD45RB and
PE-conjugated anti-CD4 mAbs and fractionated into
CD4+CD45RBhigh and
CD4+CD45RBlow fractions
under sterile conditions by two-color sorting on a FACSVantage (Becton
Dickinson). For isolation of
CD4+CD25+ T cells, purified
splenic CD4+ T cells were stained with either
anti-CD4-PE and anti-CD25-FITC or anti-CD25-FITC alone.
CD4+CD25+ subpopulation
(
10% of CD4+ T cells) were then sorted on a
FACSVantage. All populations were 99.0% pure on reanalysis.
Establishment of experimental colitis and Ab treatment
SCID mice were reconstituted by i.p. injection of syngeneic CD45RBhigh-CD4+ T cells either alone or cotransferred with syngeneic CD45RBlow-CD4+ or CD25+CD4+ cells (4 x 105/mouse of each cell population). Colitic SCID mice, reconstituted with syngeneic CD45RBhighCD4+ T cells from spleen of normal mice, were treated i.p. with either anti-B7.1 or anti-B7.2 or a combination of both mAbs or anti-CTLA-4 at the dose of 250 µg/mouse twice a week starting at the beginning of T cell transfer up to 8 wk. Mock-treated controls received similar injections of HIg and rat IgG2a on the same schedule. All mice were monitored weekly for weight, soft stool or diarrhea, and rectal prolapse. All mice were sacrificed 8 wk after T cell transfer or when they exhibited a loss of >20% of original body weight. Colonic tissues were obtained for histologic and cytologic examination.
Histologic examination and immunohistochemistry
Intestinal tissue samples were fixed in PBS containing 10% neutral-buffered formalin. Paraffin-embedded sections (5-µm) were cut and stained with H&E. The sections were analyzed without prior knowledge of the type of T cell reconstitution or treatment. Microscopic sections were graded according to the number and severity of lesions. The mean degree of inflammation in the colon was assessed using a scoring system as described previously (36).
Colonic samples for immunohistochemistry were embedded in OCT compound and snap-frozen in liquid nitrogen. The frozen samples were stored at -80°C until processed. Five-micrometer cryostat sections of colonic tissue were cut and stained for the presence of B7.1, B7.2, CD4, and F4/80 using an avidin-biotin-peroxidase complex technique as previously described (36). Briefly, serial sections were incubated for 30 min with 5 µg/ml rat anti-mouse B7.2. The rabbit anti-rat IgG (Dako, Glostrup, Denmark; 1:400) was chosen as second Ab. After three washes with PBS, the avidin-biotin-peroxidase complex (Dako) was added, and sections were incubated for 30 min. Staining for B7.1, CD4, or F4/80 Ag was performed using either biotinylated anti-B7.1 or anti-CD4 or anti-F4/80 as primary Ab at a concentration of 5 µg/ml, followed by incubation with the avidin-biotin-peroxidase complex. The color reaction was developed with 0.05% 3-amino-9-ethylcarbazole (Janssen, Beerse, Belgium). The slides were counterstained with hematoxylin. Negative controls were obtained by incubating sections with irrelevant isotype-matched HIg or rat IgG2a, or by omitting the primary Ab.
Cell preparation and cytokine analysis
Colonic lamina propria CD4+ T cells were
isolated using a method as described previously (36). For
induction of cytokine production by lamina propria
CD4+ T cells, 96-well culture plates (Nunc,
Roskilde, Denmark) were precoated with anti-CD3 (final
concentration, 5 µg/ml) in 100 µl PBS at 37°C for 4 h and
washed with PBS three times to remove unbound Ab.
CD4+ T cells (5 x
105/ml) were incubated in the presence of coated
anti-CD3 and mitomycin C-treated (50 µg/ml for 30 min at
37°C) mouse mastocytoma P815 cells (5 x
105/ml) transfected with mouse CD80 at 37°C in
5% CO2 humidified air. Samples were performed in
triplicate or quadruplicate in a total volume of 200 µl/well. After
48 h of culture, supernatants were harvested and assayed for
IFN-, IL-2, and IL-4 by ELISA as described previously
(36).
Statistical analysis
Data were statistically analyzed using Student’s t test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the role of the CD28-B7 costimulatory pathway in the
induction of intestinal mucosal inflammation,
CD45RBhighCD4+ T cells from
the WT and CD28-/- mice were isolated and
reconstituted into SCID recipients (4 x
105/mouse). After an 8-wk period of observation,
SCID mice reconstituted with
CD28-/-CD45RBhighCD4+
T cells appeared healthy and none of them developed wasting disease
with colitis, as evidenced by gradual increase of body weight (Fig. 1
A) and absence of diarrhea
and prolapse. Histological analysis of colonic sections revealed normal
mucosa of colon with absence of inflammatory cell infiltrates (Fig. 1B), being indistinguishable from that in naive SCID mice.
In contrast, WT CD45RBhigh cell-reconstituted
mice developed severe colitis. Histologic analysis showed transmural
inflammation with large numbers of leukocyte infiltrates in colon (Fig. 1C), similar to our earlier report (36). Lower
numbers of CD4+ T cells were recovered from the
colon of
CD28-/-CD45RBhigh
cell-reconstituted mice (0.24 ± 0.03 x
106/colon, n = 12) as compared
with those from WT cell-reconstituted mice (2.11 ± 0.51 x
106/colon, n = 8)
(p < 0.001). Immunohistochemical analysis also
demonstrated few CD4+ T cells and macrophages in
the lamina propria in
CD28-/-CD45RBhigh
cell-reconstituted mice (data not show). In additional experiments,
SCID mice were also reconstituted with either
CD28-/-CD45RBlowCD4+
T cells (n = 5) or cotransferred with both
CD28-/-CD45RBhigh and
CD28-/-CD45RBlow cells
(n = 5). None of these cell subsets was able to induce
mucosal inflammation after an 8-wk observation. The data show that
activation of
CD45RBhighCD4+ T cells and
induction of mucosal inflammation are dependent on the CD28-B7
costimulatory pathway.
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In situ expression of B7.1 and B7.2 in the inflamed colon was
studied by immunohistochemistry. Colonic samples were obtained from
colitic SCID mice 8 wk after reconstitution with syngeneic WT
CD45RBhighCD4+ or
CD45RBlowCD4+ T cells
(36). Fig. 2 shows that the
number of B7.1- and B7.2-positive cells was markedly increased in the
inflamed mucosa. The majority of positive cells were located in the
lamina propria and submucosa, but some in the tunica muscularis and
subserosa. The positive cells were mostly diffusely distributed, but
also formed aggregates, suggesting a granulomatous type of
inflammation. Intestinal epithelial cells were negative for B7. In
contrast, only weak expression of B7.1 and B7.2 was observed in the
colonic tissue from SCID mice reconstituted with
CD45RBlowCD4+ T cells (Fig. 2, C and D). These results show that B7 molecules
are overexpressed in inflamed mucosa of colitic SCID mice and support
the concept that B7 may participate in the immunopathology of
colitis.
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After confirming increased expression of B7.1 and B7.2 in the
inflamed mucosa of colitic SCID mice, we asked whether blockade of the
B7 costimulatory signaling could interfere with the development of
intestinal mucosal inflammation. SCID mice reconstituted with syngeneic
WT CD45RBhighCD4+ T cells
were treated with i.p. injection of either anti-B7.1, anti-B7.2
mAb, a combination of both Abs, or control HIg or rat IgG2a at the
dosage of 250 µg/mouse twice weekly starting at the beginning of T
cell transfer and continuing up to 8 wk. HIg- or rat IgG2a-treated mice
developed wasting disease with severe colitis and loss of body weight
(Fig. 3A), soft stool or
diarrhea with increased mucus, and anorectal prolapse. These mice had
an enlarged colon with a significantly thickened wall. Transmural
inflammation with increased leukocyte infiltrates was commonly seen in
the ascending and transverse colon. Epithelial lesions included loss of
goblet cells, crypt abscesses, and extensive ulceration (Fig. 4A). Moreover, architectural
changes such as crypt elongation, villous transformation of the
surface, and crypt branching were also observed. Anti-B7.2-treated mice
still developed colitis with comparable severity, time course, and
histological scores to control IgG-treated recipients (Figs. 3A and 4B). In contrast, anti-B7.1-treated
mice appeared healthy with gradual increase of body weight and absence
of diarrhea (Fig. 3A). Histological features of colon were
almost normal (Fig. 4C), and histological scores were
significantly decreased compared with those in HIg-treated mice (Fig. 3B). In addition, a mixture of mAb to B7.1 and B7.2 also
prevented wasting disease with colitis, being indistinguishable from
anti-B7.1 alone (Fig. 3A). Histological analysis of
colonic tissue revealed normal mucosa, similar to findings in Fig. 4C (data not shown).
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Colonic lamina propria CD4+ T cells were isolated
from the colonic samples of colitic SCID mice treated with either
anti-B7.1 or anti-B7.2 or controls. CD4+
T cells (5 x 105/ml) were incubated in the
presence of coated anti-CD3 (5 µg/ml) and mouse mastocytoma
P815 cells transfected with mouse CD80 (5 x
105/ml) at 37°C in 5%
CO2 humidified air. Fig. 3
, C and
D show that IFN- and IL-2 were significantly reduced in
the supernatants of CD4+ T cell cultures from
anti-B7.1-treated mice compared with those from either
anti-B7.2- or control IgG-treated recipients
(p < 0.005). IL-4 was found to be at or below
the detection limit of the assay. Thus, anti-B7.1 treatment
down-regulates proinflammatory cytokine secretion without evidence for
induction of a Th2 shift. Moreover, the data indicate that B7.1 is the
dominant costimulatory molecule that potentiates lamina propria T cell
activation and differentiation during the induction of colitis, similar
to what has been reported in EAE (11, 12, 13).
Anti-CTLA-4 exacerbates the development of colitis
Colitic SCID mice were then injected i.p. with either
anti-CTLA-4 or HIg at the dose of 250 µg twice a week starting at
the time of T cell transfer. Because our preliminary experiments had
demonstrated that anti-CTLA-4-treated mice developed severe colitis
after 6-wk administration, both anti-CTLA-4- and HIg-treated mice
were sacrificed after 6 wk. The clinical manifestations during
treatment were monitored as described above. HIg-treated mice at that
time point had a slight decrease of body weight and soft stool (Fig. 5A). Diarrhea with increased
mucus was observed in two of eight mice. Histological analysis of
colonic sections revealed that HIg-treated mice had already developed
an active mild colitis with leukocyte infiltration in the lamina
propria, occasionally in the submucosa and the muscular layers, and
absence in the serosa (Fig. 6A). Leukocyte infiltration
was characterized by focal aggregation at the apical areas of villi and
the basal areas, or by diffuse infiltration in the lamina propria.
Epithelial lesions included mucin depletion, crypt branching, crypt
elongation, and transformation. Ulceration was not found in all
samples. In contrast, anti-CTLA-4-treated mice started progressive
weight loss already 2–3 wk after T cell transfer (Fig. 5A).
By 6 wk they had severe wasting disease with colitis, diarrhea with
increased mucus in the stool, marked decrease of body weight
(p < 0.05), and anorectal prolapse. Colonic
sections revealed transmural inflammation with significantly increased
leukocyte infiltration in any part of the colon from all mice.
Epithelial lesions included mucin depletion, loss of goblet cells,
crypt abscesses, and ulceration, occasionally with a mountain-peak
appearance (Fig. 6B). The mucosal abnormalities were also
seen in the ileocecum. Histological scores of colonic samples were
significantly increased in anti-CTLA-4-treated mice (6.5 ±
2.4) compared with HIg-treated mice (3.1 ± 1.5)
(p < 0.01). The CD4+ T
cell recovery from the inflamed colon was also markedly increased in
these mice (2.45 ± 0.84 x 106)
compared with that from the HIg-treated recipients (1.02 ±
0.36 x 106) (p <
0.01). Additionally, splenomegaly with large numbers of lymphocyte
infiltrates in the white pulp was commonly seen (data not shown).
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(5 µg/ml) and mouse CD80-transfected P815 cells
(5 x 105/ml) for 48 h at 37°C in 5%
CO2 humidified air. CD4+ T
cells from anti-CTLA-4-treated mice produced more IFN- and IL-2
compared with controls (p < 0.01, Fig. 5, B and C). IL-4 and IL-5 were slightly increased
in the culture supernatants but with no significant difference between
these two groups (data not shown). These data indicate that
anti-CTLA-4 treatment results in an accelerated onset of disease,
an increased severity of colitis, and an up-regulated Th1 immune
response. CD25+CD4+ regulatory T cells function in a CD28-independent way
CD45RBlowCD4+ T cells
do not induce colitis but are able to prevent colitis development if
cotransferred with WT CD45RBhigh cells into SCID
mice (36). More recently, it has been reported that
CD25+CD4+ cells among the
CD45RBlowCD4+ T cells
function as the regulatory T cells to inhibit intestinal inflammation
(38, 42) and other experimental autoimmunity models
(43), and that regulatory T cell activity depends on IL-10
secretion (44). Therefore, we addressed the question
whether these regulatory T cells also require CD28 costimulation. This
was studied by cotransfer of CD45RBlow or
CD25+CD4+ T cells from
CD28-/- mice together with disease-inducing WT
CD45RBhighCD4+ T cells. As
shown in Fig. 7, A and
B, cotransfer of WT CD45RBlow or
CD25+CD4+ T cells
effectively inhibited wasting disease with colitis over an 8-wk period
of observation. No detectable pathological changes were observed in the
bowel wall, consistent with earlier reports (36, 38). Of
particular note, cotransfer of CD45RBlow or
CD25+CD4+ cells from
CD28-/- mice still effectively suppressed
experimental colitis in SCID recipients within the same observation
period, as evidenced by the gradual increase of body weight and absence
of diarrhea. The colon appeared normal when animals were sacrificed.
Histological analysis of colonic sections illustrated only few
leukocyte infiltrates in the lamina propria. Transmural inflammation
and epithelial hyperplasia were effectively inhibited. Histological
scores were significantly decreased compared with those in WT
CD45RBhigh cell-reconstituted mice
(p < 0.01), but not different from those in
SCID mice cotransferred with WT CD45RBlow or
CD25+CD4+ cells (Fig. 7C). Colonic lamina propria CD4+ T
cell recovery from mice cotransferred with either
CD45RBlow or
CD25+CD4+ cells was also
decreased compared with that from recipients reconstituted with WT
CD45RBhigh cells alone (p
< 0.01, Fig. 7D). Moreover, lamina propria
CD4+ T cells, when stimulated with Con A (1
µg/ml), also secreted lower levels of IL-2 and IFN- compared with
those from WT
CD45RBhighCD4+
cell-reconstituted mice (data not shown). These findings indicate that
the regulatory functions of
CD45RBlowCD4+ and
CD25+CD4+ T cells are
CD28 independent.
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CTLA-4 was reported to be constitutively expressed on a subset of
regulatory CD25+CD4+ T
cells (38, 42). We also analyzed expression of CTLA-4 on
freshly isolated CD25+CD4+
T cells and found that
CD25+CD4+ T cells from
CD28-/- mice expressed comparable levels of
CTLA-4 as the WT subsets (data not shown). To further illustrate
the role of B7-CTLA-4 interaction in the effector functions of
these regulatory T cells, WT
CD45RBhighCD4+
cell-reconstituted SCID mice were cotransferred with
CD25+CD4+ cells from either
WT or CD28-/- mice, and they were treated with
either anti-CTLA-4 or HIg. As indicated in Fig. 8, after 8 wk of administration,
HIg-treated mice appeared healthy as evidenced by gradual increase of
body weight and absence of diarrhea. No detectable pathological changes
were seen in the colon. In contrast, all of anti-CTLA-4-treated
mice developed wasting disease with colitis. Histological analysis of
the colon revealed the same features as shown in Fig. 4A.
Therefore, the data, together with previous reports (38, 42), substantiate the critical role of B7-CTLA-4 interaction for
the functioning of the regulatory T cells that control inflammatory
responses in the gut.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2) by lamina propria
CD4+ T cells. In contrast, anti-CTLA-4
treatment deteriorated mucosal immune responses with enhanced
inflammatory damage in the intestine and up-regulation of local
proinflammatory cytokine secretion. Of note,
CD45RBhighCD4+ T cells from
CD28-/- mice failed to induce intestinal
mucosal inflammation after transfer into SCID recipients.
CD45RBlow and
CD25+CD4+ T cells from
CD28-/- mice, similar to the WT subsets, still
prevented severe colitis induced by reconstitution with WT
CD45RBhigh cells of SCID mice. Therefore, our
findings provide evidence that the CD28-B7.1 costimulatory pathway
plays an important role in promoting pathogenic
CD4+ T cell activation and differentiation and
that CTLA-4-B7 interaction delivers a negative signal to prevent or
down-regulate inflammatory responses in the intestine. Moreover, these
data also implicate that CD28-B7 interaction is not required for the
effector functions of regulatory T cells.
Colitis in this model is dependent on the transfer of
CD45RBhighCD4+ cells, which
home after activation to the gut wall where further activation leads to
proinflammatory cytokine production.
CD28-/-CD45RBhighCD4+
T cells failed to cause intestinal inflammation when reconstituted into
SCID recipients. These findings indicate that the induction of effector
response of this subset of T cells and their homing to the lamina
propria is CD28 dependent. Previous work has shown that
CD28-/- T cells have decreased proliferative
responses to Ags and costimulatory stimulation and produce low levels
of IL-2 (4, 5). CD28-/- mice have
a deficient T cell activation, as evidenced by the findings that they
exhibit delayed cardiac allograft rejection after transplantation
(23) and are resistant to collagen-induced arthritis with
low IFN- secretion by lymph node cells in response to collagen
stimulation (21). Requirement of CD28 for disease
induction might depend on interaction with either B7.1 or B7.2 or both.
In the present study, B7.1 and B7.2 were both highly expressed in the
inflamed mucosa of colitic SCID mice. We suggest that this increase in
expression of B7.1 and B7.2 is induced either directly by luminal
bacterial products or by activated lamina propria T cells via CD40-CD40
ligand interaction (36, 39). Anti-B7.1 but not
anti-B7.2 effectively prevented mucosal inflammation and
significantly down-regulated proinflammatory cytokine secretion (e.g.,
IL-2, IFN-) by lamina propria CD4+ T cells and
leukocytic infiltrates in the intestine. Thus, B7.1 appears to be
predominantly involved in the Th1-mediated mucosal inflammation in this
adoptive transfer model of colitis, which is consistent with some
previous reports suggesting a predominant involvement of B7.1 in
Th1-mediated pathology. B7.1 is generally not detected on resting APC,
whereas low levels of B7.2 are constitutively expressed on macrophages
and dendritic cells in naive mice. Upon cell activation, both B7.1 and
B7.2 are up-regulated, although expression of B7.1 often follows that
of B7.2 (3). The slower rate of dissociation of B7.1
binding to CD28, compared with B7.2, may enable it to provide more
potent costimulatory functions (45). Moreover, the
differences in B7.1 and B7.2 binding determinants of CD28 also raise
the possibility that these molecules play a distinct role in T cell
effector response (46). Some previous work has suggested
that B7.1 is preferentially involved in the Th1 immune response, while
B7.2 regulates the development of Th2 cells (11, 12, 13, 15, 16). However, not all data support such a conclusion. Anti-B7.2
but not anti-B7.1 effectively prevents the onset of diabetes but
not insulitis in nonobese diabetic mice (14). Moreover,
mice lacking either B7.1 or B7.2 are still susceptible to EAE induction
(22). These conflicting data may be attributed to several
factors, such as a strain dependency in the susceptibility of animals
and in the development of immune responses. Moreover, we cannot rule
out the possibility that differences in the affinity or in the
catabolism of both Abs could be responsible for the different outcome
of in vivo treatment. The same anti-B7.2 mAb, which was ineffective
in the present model, has been previously shown to suppress the onset
of diabetes and experimental allergic asthma (14, 15, 16).
Therefore, the respective roles of B7.1 and B7.2 in the induction of
mucosal immune responses still need to be further investigated, and in
vivo studies using recipients deficient for B7 single molecules seem
warranted.
Lack of B7-CD28 interaction prevents disease induction in this model.
The potential sequence of events by which CD28-B7 costimulation is
involved in induction of mucosal immunopathology is likely at an early
stage of the disease. Once CD45RBhighCD4 cells
are transferred into SCID mice, interaction in gut-draining lymph nodes
with APC (i.e., dendritic cells) involving CD28-B7 interactions leads
to initial triggering with low levels of IL-2 secretion
(3), up-regulation of IL-12R
2 expression
(47), and polarization toward Th1 differentiation.
CD4+ T cells, especially after CD28
cross-linking, also express high levels of CD40 ligand, which interacts
with CD40 expressed on APC to promote IL-12 secretion (36)
and B7 expression. Furthermore, IL-12 may act synergistically with B7.1
to abrogate mucosal T cell tolerance and to induce intestinal
inflammation (48). According to recent report
(49), this priming occurs in regional Ag-draining lymph
nodes and will be followed by homing of the Ag-primed cells to the
lamina propria of the gut. After priming and homing to the gut, B7
molecules might still play a role in further enhancing and/or
prolonging T cell activity. Increased IL-12R2 expression on lamina
propria T cells after B7 signal triggering has been observed in
inflamed mucosa of Crohn’s disease (50). We and others
have also reported increased expression of B7.1 and B7.2 molecules in
the inflamed mucosa of IBD patients (9, 10). Further
elucidation of the role of these molecules in the persistence of
colitis is still unclear and would require delayed treatment after
disease onset.
In addition to CD28 and its interaction with B7, we also studied the involvement of the CTLA-4 molecule. Blockade of the CTLA-4 signal with neutralizing mAb precipitated the development of intestinal mucosal inflammation induced by CD45RBhighCD4+ T cells. This was characterized by an accelerated onset of wasting disease, an increased severity of colitis, and an up-regulated secretion of Th1 proinflammatory cytokines. These results are consistent with earlier reports, showing that anti-CTLA-4 deteriorates clinical disease and augments T cell immune responses in other experimental diseases including EAE (33, 34), autoimmune diabetes (35), and allograft rejection (23). Blockade of CTLA-4 signaling with mAb promotes T cell expansion and enhances both Th1- and Th2-type effector responses (25, 29). After T cell transfer and development of colitis, CTLA-4 expression on lamina propria T cells was clearly increased (data not shown), providing a target for the effect of anti-CTLA-4. Because anti-CTLA-4 treatment resulted in the deterioration of colitis, the CTLA-4 expressed on these pathogenic cells can to some degree regulate functional responsiveness of effector T cells in vivo. The most likely mechanism by which anti-CTLA-4 deteriorates mucosal inflammation is that it directly blocks negative signals for effector T cells. Whether these effects occur at the level of priming in lymph nodes or after migration to the lamina propria is still unclear. Our findings are different from a recent report (38) showing that administration of anti-CTLA-4 to mice reconstituted with WT CD45RBhigh cells alone does not affect the development of colitis. The discrepancy could be attributed to the dosage and schedule of Ab administration as well as the genetic background of animals used.
CD25+CD4+ T cells have been
identified as important regulatory T cells, also in experimental
colitis, and they represent a subset of
CD45RBlowCD4+ T cells.
Whether these cells act at the initiation of T cell priming or rather
suppress effector T cells after homing to the gut is still unknown. In
this adoptive transfer model of colitis, the immune suppression of
CD45RBlowCD4+ cells is
dependent on IL-10 and TGF- (44, 51). Therefore, we are
interested in the question of whether these regulatory T cells require
CD28 and/or CTLA-4 for their suppressive activity.
CD45RBlow or
CD25+CD4+ T cells from
CD28-/- mice suppressed the induction of
intestinal inflammation by WT CD45RBhigh cells.
These data implicate that the effector functions of these regulatory T
cells in mucosal immune response are independent of CD28 costimulatory
signaling, consistent with a recent report (42). The
inhibitory effect of CTLA-4 engagement has also been observed in
CD28-/- T cells by others (23, 52), suggesting that CTLA-4 functions as a negative regulator of
immune responses independently of CD28.
Interaction of CTLA-4 on these regulatory T cells with B7 on APC may
provide a sufficient costimulatory signal for their expansion and their
activity (53), leading to the down-regulation of
intestinal inflammation. However, whether and how CTLA-4-B7 interaction
affects the effector function of these regulatory T cells is in fact
not yet clearly understood. Recent work reported that CTLA-4 is
constitutively expressed at high levels on
CD25+CD4+ T cells and that
CTLA-4 might be involved in the activity of these cells (38, 42). We also observed increased expression of CTLA-4 on both
freshly isolated CD45RBlow and
CD25+CD4+ T cells.
Interestingly, blockade of CTLA-4 signaling and of TGF- reversed the
suppressive effects of
CD25+CD4+ cells
(38), and regulatory T cells from
IL-10-/- mice were unable to prevent colitis
(44). CTLA-4 cross-linking leads to
CD4+ T cell TGF- production (54),
suggesting that TGF- may mediate the inhibitory effects of CTLA-4
signaling. TGF- is a pleiotropic cytokine with generally
antiinflammatory and immunosuppressive properties. Taken together,
these data suggest that TGF- and IL-10 secretion by regulatory T
cells after CTLA-4 ligation plays a role in their effector functions.
In our work, anti-CTLA-4 treatment effectively reversed the
activity of regulatory
CD25+CD4+ or
CD45RBlowCD4+ T cells. The
most obvious explanation is that anti-CTLA-4 abrogates a
B7-dependent costimulatory signal for activation for these regulatory T
cells. However, it is also possible that in vivo administration of
anti-CTLA-4 directly eliminates regulatory T cells or interferes
with their activity in another way, because triggering of CTLA-4 has
been previously shown to induce T cell apoptosis (28).
In conclusion, our findings presented here indicate that CD28-B7.1 costimulatory pathway preferentially participates in breaking tolerance and induction of mucosal immune responses in colitic SCID model. In contrast, CTLA-4 triggering seems to play an essential role in maintenance of tolerance against luminal Ags. These data may shed some light on the pathogenic mechanisms involved in human IBD, where the role of regulatory T cells should be further investigated. Also the exact role of B7.1 and B7.2 in the pathogenesis of IBD needs to be further evaluated, eventually by targeting with mAb. Such experiments might increase the insight into the immunoregulatory mechanisms in the intestine and might provide a new therapeutic approach in human IBD.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan L. Ceuppens, Laboratory of Experimental Immunology, U.Z. Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail address: Jan.Ceuppens{at}med.kuleuven.ac.be
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; EAE, experimental autoimmune encephalomyelitis; HIg, hamster IgG; WT, wild type; KO, knockout.
Received for publication January 11, 2001. Accepted for publication May 30, 2001.
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