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*
Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4
7. Most importantly,
the anti-CD134L mAb completely blocked development of colitis.
Surprisingly, CD134L was found to be expressed by a proportion of
dendritic cells (DC) in the MLNs of unreconstituted SCID mice,
suggesting that CD134L can be induced on DC in the absence of T
cell-derived signals. These results indicate that some DC in the MLNs
of SCID mice express an activated phenotype and that CD134L expression
by these cells is involved in the development of colitis induced by T
cell transfer. Accumulation of CD134L+ DC was inhibited by
cotransfer of regulatory T cells, suggesting that inhibition of the
accumulation of activated DC is one mechanism by which these cells
prevent immune pathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfer of small numbers of CD45RBhigh
CD4+ T cells to SCID mice led to significant
expansion of these cells in the periphery. This was most marked in the
intestine, as the majority of mice developed a Th1 cell-mediated
chronic colitis (7, 8, 9, 10, 11). Intestinal pathology resembled
that seen in inflammatory bowel disease
(IBD)5 in humans and
was characterized by an extensive lymphocytic infiltrate,
comprising CD4+ T cells and macrophages,
epithelial cell hyperplasia, ulceration, and depletion of
mucin-secreting goblet cells (12). T cell expansion was
significantly reduced and intestinal pathology absent when T cells were
transferred to SCID mice raised under germ-free conditions, indicating
that the indigenous microbiota play an important role in driving these
pathogenic responses (13, 14). Importantly, cotransfer of
the reciprocal CD45RBlow subset inhibited both
the dysregulated expansion of CD45RBhigh progeny
and the development of colitis by a mechanism that involved both IL-10
(15) and TGF- (16). Recently, these
regulatory T (Treg) cells have been shown to be contained within the
CD25+ CD45RBlow
CD4+ subset and to be dependent on CTLA4 for
their function (17). CD25+
CD4+ cells have also been shown to prevent the
development of autoimmune disease induced after T cell depletion,
indicating that functionally specialized Treg cells, present in the
Ag-experienced pool of normal mice, play an important role in the
control of peripheral T cell responses (18, 19, 20).
It is now widely accepted that T cell activation involves signals transduced by the TCR complex after recognition of Ag as well as from costimulatory molecules after encounter with their ligands present on APC (21). Members of the TNFR superfamily, including CD27, CD30, 4-1BB (CD137), and OX40, have been shown to mediate costimulatory activity (22). The OX40 molecule (CD134) is expressed transiently on activated CD4+ T cells (23, 24) and on some CD8+ cells. It interacts with OX40L (CD134L) (25, 26), which is a type II membrane protein with amino acid sequence similarity to TNF. CD134L has been reported to be present after activation on B cells (27, 28), dendritic cells (DC) (29), microglia (30), and human endothelium (31). Studies in both the mouse and human indicate that CD134-CD134L interactions are required for optimal B cell responses (27, 32, 33). More recent studies have revealed that these interactions also play a pivotal role in T cell responses. Activation of T cells in the presence of CD134L-expressing cells led to enhanced clonal expansion and secretion of cytokines (24). Similar results have been observed in vivo, as treatment of mice with anti-CD134 mAb in the presence of LPS led to enhanced clonal expansion and development of T cell memory (34). Consistent with these findings, CD134 knockout mice were found to have reduced T cell proliferative responses and cytokine production defects (35, 36). Similarly, CD134L knockout mice exhibited impaired T cell priming and cytokine production in vivo. Furthermore, DC isolated from these mice had impaired costimulatory activity in vitro, indicating that CD134L expression plays an important role in APC function. (37, 38).
Several studies have highlighted the importance of CD134-CD134L interactions in vivo. Thus, CD134+ T cells are found in inflammatory lesions, and blocking CD134-CD134L interactions via administration of a CD134-Ig fusion protein led to amelioration of experimental autoimmune encephalomyelitis (EAE) (30) and of intestinal inflammation (30, 39, 40). Although these studies clearly show that CD134-CD134L interactions play a role in T cell-mediated immune pathology, they do not reveal precisely how. The fact that CD134L is expressed on a variety of activated APC as well as on activated endothelium makes it a possibility that CD134-CD134L interactions are involved in a range of T cell functions, including expansion and survival, differentiation and effector function, as well as migration to sites of inflammation.
Given the important role that CD134-CD134L interactions play in the function of T cells in an intact immune system, it was a possibility that these interactions were involved in T cell-mediated immune pathology induced under conditions of lymphopenia. To test this, we have generated a mAb reactive with murine CD134L. Using this reagent in SCID mice restored with CD45RBhigh CD4+ T cells, we show that CD134-CD134L interactions are essential for dysregulated T cell expansion and development of colitis, and that it is expression of CD134L by activated DC that drives the pathogenic response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C.B-17 SCID, 129/SvEv recombinase-activating gene (Rag) 2-deficient (Rag2-/-), BALB/cJ, and 129/SvEv mice were bred under specific pathogen-free conditions and kept in microisolators with filtered air in the Biomedical Service Unit at the John Radcliffe Hospital. Mice were used at 8–12 wk of age.
Antibodies
The following mAbs were used for cell purifications: YTS169,
anti-mouse CD8; TIB120, anti-mouse MHC class II (American Type
Culture Collection (ATCC), Manassas, VA); M1/70, anti-mouse Mac-1
(TIB128; ATCC); RA3-6B2, anti-mouse B220 (41);
PE-conjugated anti-mouse CD45RB (clone 16A; PharMingen, San Diego,
CA); Cy-Chrome-conjugated anti-mouse CD4 (clone RM4-5; PharMingen).
The following Abs were used for flow cytofluorography: TIB 139,
anti-mouse H-2b, CD4-PerCP (RM4-5), CD45RB FITC (16A),
47 PE (LPAM-7),
E-bio (M290), CD25-bio (7D4), CD11c PE (HL3),
CD40 FITC (HM40-3), CD80 FITC (16-10A1), CD86 FITC (GL1), SAv-APC (all
PharMingen), and CD134 FITC (OX86) (Serotec, Oxford, U.K.). Stained
cells were run on a FACSort and analyzed using CellQuest Software
(Becton Dickinson, San Jose, CA).
OX89, a rat IgG1 anti-OX40L mAb, was produced by standard procedures involving the fusion of NS1 myeloma cells and spleen cells from a PVG rat that had been immunized, s.c., with 20 µg CD4.CD134L (see below) in complete Freund’s adjuvant. After 2 wk, the rats received CD4.CD134L (20 µg) in incomplete Freund’s adjuvant, and after a further 3-wk interval they received 20 µg of CD4.CD134L i.v. Splenocytes were taken 4 days after the last immunization. Initial screening was on recombinant protein, and activated T cells and specificity were confirmed on fibroblasts transfected with rat OX40L (24). For in vivo use, anti-mouse CD134L (OX89) and the rat anti-mouse IgG1 isotype control (GL113) were purified from hybridoma supernatant by affinity chromatography and shown to contain <1 EU endotoxin per mg of protein.
Blocking studies
Soluble (s)CD4.CD134 protein contains the TNFR-like repeat region of rat CD134 that binds to mouse CD134L plus domains 3 and 4 of rat CD4, and was produced and purified, as described (26). A multimeric form of the protein was generated by binding CD4.CD134 recombinant protein to streptavidin-coated fluorescent Sphero beads (Spherotech, Libertyville, IL) via a biotinylated OX68 Ab, as described previously (42, 43). The cells and Sphero beads were incubated on ice for 40 min and analyzed by flow cytofluorography (42, 43). The chimeric sCD48.CD4 protein contained domains 3 and 4 of CD4 as for CD134 (42).
Cell purification and flow cytofluorography
CD4+ T cell subsets were isolated from spleens, essentially as previously described (9). Briefly, single cell suspensions were depleted of CD8+, MHC class II+, Mac-1+, and B220+ cells by negative selection using sheep anti-rat-coated Dynabeads (Dynal, Oslo, Norway). In some cases, CD4+-enriched cells were stained with Cy-Chrome-conjugated anti-CD4 and PE-conjugated anti-CD45RB Abs and CD4+ CD45RBhigh and CD45RBlow CD4+ fractions sorted on a FACSVantage (Becton Dickinson). Alternatively, cells were stained with FITC-conjugated anti-CD45RB, PE-conjugated anti-CD25, and CyChrome-conjugated anti-CD4, and CD45RBhigh CD4+and CD25+ CD4+ populations were sorted. Cells were >97% pure on reanalysis. In some experiments, CD4+ T cells were purified by positive selection using CD4 Dynabeads and the DETACHaBEAD system (Dynal), followed by MACS separation to yield CD45RBhigh CD4+ cells. For the MACS separation, CD4+ cells were stained with FITC-conjugated anti-CD45RB (16A; PharMingen), followed by anti-FITC microbeads, according to the manufacturer’s instructions, and run over MS+ mini columns (Miltenyi, Bergisch Gladbach, Germany). Bound cells were eluted from the column and were 95% CD45RBhigh CD4+.
CFSE labeling of T cells
T cell division in vivo was assessed by flow cytofluorography of CFSE-labeled cells. MACS-sorted CD45RBhigh CD4+ cells were stained in vitro with the cytoplasmic dye CFSE (Molecular Probes, Leiden, The Netherlands) before reconstitution (44). Briefly, cells were incubated for 7 min at 37°C with 5 µM CFSE. The reaction was quenched by washing in ice-cold DMEM supplemented with 10% FCS. Cell viability was assessed by trypan blue exclusion. CFSE staining gave one sharp peak, as assessed by flow cytofluorography.
T cell reconstitution and Ab treatment
Eight- to 10-wk-old C.B-17 SCID mice were injected i.p. with 4 x 105 sorted CD45RBhigh CD4+ cells alone or in combination with CD45RBlow CD4+ or CD25+ CD4+ Treg cells, as indicated. The 129 Rag2 -/- mice were reconstituted with 2.5 x 106 CFSE-labeled MACS-sorted CD45RBhigh CD4+ cells. mAbs (500 µg) were injected i.p. in PBS the day after reconstitution and twice per week for the duration of the experiment.
Immunohistochemistry
Frozen tissue was sectioned and fixed in 2% formaldehyde in PBS. Endogenous peroxidases were neutralized by incubation with 1% H2O2. The primary Abs were used at a concentration of 5 µg/ml (OX89) or as unpurified tissue culture supernatant (N418). The secondary Ab was a biotinylated anti-rat or anti-Armenian hamster. Positive brown staining was developed by the ABC-elite kit, followed by diaminobenzidine substrate (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin.
Cell purification
For enumeration of CD4 cells in the intestine lamina propria, lymphocytes were isolated from the colon, as described (15). The total number was determined by multiplying the number of leukocytes by the frequency of CD4+ cells. The latter was determined by flow cytofluorography. DC were prepared essentially as in Vremec and Shortman (45). Briefly, mesenteric lymph nodes (MLNs) were cut into pieces and incubated for 25 min under agitation at 37°C in the presence of 1 mg/ml collagenase/dispase (Sigma, St. Louis, MO) and 100 U/ml DNase (Sigma) before 5 min of deaggregation in the presence of 0.1 M EDTA. The tissue was then passed through a 70-µm membrane to generate single cell suspensions.
Clinical and microscopic examination
T cell-restored SCID mice were weighed weekly and sacrificed after 8 wk or when they had lost 20% of their initial weight. A 0.5-cm piece of the distal colon was removed and fixed in formal saline. Paraffin-embedded sections (5 µm) were cut and stained with hematoxylin and eosin and used for microscopic assessment of colitis. Tissues were graded semiquantitatively from 0 to 5 in a blinded fashion. A grade of 0 was given when there were no changes observed. Changes typically associated with other grades are as follows: grade 1, minimal scattered mucosal inflammatory cell infiltrates, with or without minimal epithelial hyperplasia; grade 2, mild scattered to diffuse inflammatory cell infiltrates, sometimes extending into the submucosa and associated with erosions, with minimal to mild epithelial hyperplasia and minimal to mild mucin depletion from goblet cells; grade 3, mild to moderate inflammatory cell infiltrates that were sometimes transmural, often associated with ulceration, with moderate epithelial hyperplasia and mucin depletion; grade 4, marked inflammatory cell infiltrates that were often transmural and associated with ulceration, with marked epithelial hyperplasia and mucin depletion; and grade 5, marked transmural inflammation with severe ulceration and loss of intestinal glands.
Statistics
The Mann-Whitney U test was used for comparison of weights, clinical scores, cell numbers, and levels of activation markers. Disease incidence was analyzed by the Fischer exact test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD134L mAb (OX89) was produced by the fusion of spleen cells
from a rat that had been immunized with a recombinant chimeric protein
consisting of domains 3 and 4 of rat CD4 and the extracellular region
of mouse CD134L. Hybridomas were screened for their ability to bind to
recombinant protein, activated T cells, and cells transfected with
recombinant mouse CD134L. As shown in Fig. 1
A, OX89 bound specifically to
CD134L-transfected fibroblast cells. Consistent with previous reports
of expression of CD134L, OX89 was also found to bind to B cells and DC
activated by LPS and anti-CD40 mAb, but not to
anti-CD3-activated T cells (data not shown). Immunohistochemical
analysis of spleens from naive BALB/c mice revealed only scattered
OX89-positive cells that were located outside the T and B cell areas
and had DC-like morphology (data not shown).
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B). Preincubation with the OX89 mAb reduced CD134 bead
binding by 
70%. Thus, these data show that OX89 binding is able to
partially block multimeric CD134 from binding to trimeric CD134L (Fig. 1C). Thus, biological effects of OX89 (see below) could be
due to blocking the interaction, although other effects such as
signaling through CD134L cannot be ruled out. OX89 treatment inhibits wasting disease and colitis
To investigate the effects of OX89 on T cell-mediated immune
pathology, C.B-17 SCID mice were reconstituted with
CD45RBhigh CD4+ T cells and
treated with either OX89 or an isotype control mAb. Weights were
followed throughout the experiments. Mice were sacrificed after 8 wk,
and the development of colonic inflammation was assessed. As expected,
the majority of control mice developed wasting disease and colitis. In
contrast, none of the anti- CD134L-treated mice had significant
inflammatory changes in the intestine (Fig. 2A) and gained weight
throughout the course of the experiment (Table I). As has been previously described,
colitis was accompanied by an expansion of CD4+ T
cells in the intestine (341 x 103 ±
89 x 103; Fig. 2B). In
OX89-treated mice, colons had to be pooled to obtain quantifiable
numbers of CD4 cells. Pools of two colons yielded 36.8 x
103 ± 11.8 x 103
CD4+ cells, meaning that there were 10- to
20-fold lower numbers of CD4 cells per colon in OX89-treated mice.
Taken together, these data demonstrate that administration of OX89
prevents T cell accumulation in the intestine of T cell-restored SCID
mice and inhibits the development of Th1-mediated colitis.
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). OX89 treatment inhibits T cell expansion and homing in T cell-restored immune-deficient mice
Colitis develops in SCID mice after transfer of as few as 104 CD45RBhigh CD4+ cells (9), and is consistently accompanied by significant numbers of CD4+ T cells in the large intestine as well as expansion in peripheral lymphoid organs. This suggests that induction of disease is dependent on initial peripheral expansion of T cells, followed by homing of these cells to the intestinal mucosa. OX89 may be acting at several points in this pathway, including inhibition of peripheral T cell reconstitution in the spleen or MLN or subsequent homing of cells to the intestine. To further investigate this, the number of T cells in different compartments early after T cell reconstitution was analyzed.
Total CD4+ T cell numbers in spleens were
determined at various time points after T cell reconstitution in
OX89-treated and control mice. As can be seen in Fig. 3A, OX89 treatment had no
effect on T cell accumulation in the spleen, as T cell numbers were
similar in both groups. To assess proliferation,
CD45RBhigh CD4+ cells were
labeled with the cytoplasmic dye CFSE and transferred to 129
Rag2-/- mice, which were treated with OX89 or
control mAb. Analysis of CFSE expression among T cells in the spleen 8
days after T cell reconstitution showed that the majority had undergone
over five divisions, as less than 10% retained detectable levels of
CFSE. This was the same in OX89- and control-treated groups (data not
shown). These results suggest that T cell accumulation in the spleen is
not dependent on CD134-CD134L interactions. However, a more extensive
kinetic analysis would be required to provide definitive evidence on
this point.
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B). Consistent with the
impaired proliferative response in OX89-treated mice, there was reduced
expression of markers of T cell activation among
CD4+ cells in the MLN, including CD25 (data not
shown).
T cell homing to the gut, including the intestine and its associated
lymphoid tissue, involves the interaction of the
47 integrin on T
cells with its ligand mucosal addressin cell adhesion molecule 1,
expressed on the vascular endothelium (46, 47). Analysis
of 47 expression on
CD4+ cells in the MLN revealed an 2-fold
reduction in the frequency of
47+
cells present in OX89 compared with isotype-treated mice (Fig. 3C). This reduction was specific for this gut-homing
molecule, as levels of
E7, thought to be
involved in retention of T cells in the gut (48), were
unchanged (data not shown). Consistent with the reduction in the
frequency of
47+
cells, there was also a striking reduction in the number of
CD4+ T cells present in the intestine early after
T cell reconstitution in OX89-treated mice (3.5 x
103 ± 0.23 x 103)
compared with control mice (18.8 x 103 ±
5.2 x 103; Fig. 3D). When lamina
propria lymphocytes were analyzed from mice reconstituted with
CFSE-labeled cells, the lamina propria lymphocytes were always CFSE
negative, indicating that cells here had undergone at least six to
eight divisions, most likely before their migration to the intestine.
These results suggest that OX89 inhibits intestinal inflammation in
part as a result of its ability to inhibit Ag-driven T cell activation
and expansion in the MLN at early time points after T cell
reconstitution. These experiments do not rule out the possibility that
OX89 also inhibits T cell expansion in the spleen or the survival or
effector function of T cells present in the intestine.
Colitis is characterized by an increase in the number of DC in the MLN that express CD134L
To further characterize where CD134+
CD4+ T cells interact with
CD134L+ cells, the expression of CD134 and CD134L
in the MLN and colon was investigated by flow cytofluorography and
immunohistochemistry. Immunohistochemical analysis of MLN revealed
significant numbers of CD134L+ cells only in mice
with colitis, with few positive cells detectable in BALB/c mice.
Significantly, there was a reduction in the number of
CD134L+ cells in mice protected from colitis by
transfer of CD45RBhigh and Treg cells (Fig. 4A) or after treatment with
OX89 (data not shown). Lack of detectable expression of CD134L in the
MLN of OX89-treated mice was not a result of blockade due to bound mAb,
as addition of a secondary anti-Rat Ig reagent failed to reveal
positive cells.
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A). In all cases,
the CD134L+ cells were CD11c positive, suggesting
that these cells are DC. They were also found to be
CD11b+ and CD8-,
suggesting that they are of myeloid origin (49).
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A, Fig. 5
legend), the cellularity of the MLN was very different between groups.
Thus, the total cell number in colitic mice ranged from 2 to 11 x
106, whereas MLN from unrestored SCID mice or
SCID mice protected from colitis by transfer of Treg cells contained
10-fold fewer cells (0.2–0.6 x 106).
Therefore, not only was there an increase in the proportion of DC that
were CD134L+, there was also a significant
increase in the total number of CD11c+ DC in the
MLN of colitic mice. As a consequence of these two factors, it follows
that there was a 24- to 36-fold (compared with SCID mice given a
mixture of CD45RBhigh CD4+
+ Treg cells) or 15-fold (compared with BALB/c) increase in the total
number of CD134L+
CD11c+ DC in these mice. Expression of CD134L on
DC is a marker of DC activation (50). So our data suggest
that there is an abnormal accumulation of activated DC in the MLN of
colitic mice. Consistent with this, CD134L+ DC
from MLN of colitic mice also expressed CD40 and CD80, both markers of
activation (data not shown). Flow cytometry also revealed detectable
numbers of CD134L+ CD11c+
DC in unmanipulated SCID mice, albeit 12-fold lower than in mice with
colitis. It should be noted that while there are detectable
CD134L+ cells in the MLN of SCID mice (Fig. 4A), these MLN are very leukopenic, meaning that the total
numbers of CD134L+ CD11c+
cells are low (Fig. 5A). The fact that mice protected from
colitis by transfer of both CD45RBhigh
CD4+ and CD25+
CD4+ Treg cells had a similar number of
CD134L+ CD11c+ DC to that
found in unreconstituted SCID mice indicates that transfer of Treg
cells prevents the accumulation of activated DC in the MLN.
Immunohistochemical analysis revealed scattered
CD11c+ cells in the lamina propria of colons from
unmanipulated BALB/c and CB.17-SCID mice as well as in mice
reconstituted with CD45RBhigh
CD4+ and CD45RBlow
CD4+ or CD25+
CD4+ Treg cells. In contrast, colitic mice,
reconstituted with CD45RBhigh
CD4+ cells only, had a very cellular lamina
propria with an abundance of CD11c+ cells
(Fig. 4B). Despite this accumulation of DC in the colon of
colitic mice, there was undetectable CD134L expression (Fig. 4B). Consistent with the lack of CD134L expression in the
colon after T cell reconstitution, there were no detectable
CD134+ CD4+ cells either
assessed by both immunohistochemistry and flow cytofluorography (data
not shown). In contrast, and a good positive control for the reagents,
CD134+ CD4+ T cells were
readily detectable in the MLN of T cell-restored SCID mice (ranging
from 5 to 30% of CD4+ T cells), but not in the
MLN of normal immune-competent mice (Fig. 5B). The frequency
of CD134+ CD4+ cells did
not correlate with colitis, as similar frequencies of positive cells
were found in the MLN of colitic mice as were found in T cell-restored
SCID mice that did not develop colitis, including OX89-treated mice and
mice that received a mixture of CD45RBhigh
CD4+ cells plus Treg cells.
Results of flow cytofluorography and immunohistochemistry suggest that interaction between CD134L+ APC and CD134+ CD4+ T cells most likely occurs in the MLN rather than in the colon of T cell-restored immune-deficient mice. These results taken together with the finding that OX89 treatment impedes T cell activation in the MLN and prevents the development of colitis suggest that T cell encounter with CD134L+ DC in the MLN is crucial for the pathogenesis of intestinal inflammation in T cell-restored SCID mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism of action of OX89 is not known, but it could act in one
or more of three possible ways: first, it could block the interaction
of CD134 and CD134L and prevent signal transmission. Although the mAb
did not block the interaction between purified recombinant proteins, it
gave about 70% inhibition in an assay designed to mimic the
multivalent interaction between cells (Fig. 1). Second, the OX89 mAb
could act by down-regulating CD134L, and indeed treated animals had few
detectable CD134L+ cells in their MLN (data not
shown). Third, it is possible that the OX89 could also give signals to
the DC, as CD134L has been shown to interact with signaling proteins
through its cytoplasmic domain (51).
CD134L is reported to be expressed by a variety of cell types after activation, including B cells, DC, and vascular endothelium (27, 28, 29, 30, 31). This makes it possible that blockade of CD134-CD134L interactions may affect both T cell priming and migration. The finding that CD134L was expressed predominantly by DC in the MLN of mice with colitis and that OX89 treatment led to retarded T cell proliferation and expression of activation Ags in the MLN, suggests that CD134L expression on DC plays an important role in driving the T cell response in this model. These data are consistent with a number of recent studies that have highlighted the important role that CD134L expression by DC plays in the development of optimal T cell responses. Mice with a targeted disruption of CD134L mounted impaired contact hypersensitivity reactions, and DC from these mice were found to be deficient in inducing T cell proliferation and cytokine secretion in vitro (37, 38). Although treatment with OX89 inhibited expression of CD134L on DC in the MLN, there was no alteration in CD134 expression on T cells in the MLN of treated vs untreated mice. As expression of CD134 is restricted to activated T cells, these data are consistent with previous findings, suggesting that CD134-CD134L interactions are important for the amplification of primed cells as opposed to their initial priming (24).
Transfer of T cells to a lymphopenic environment leads to substantial expansion of the transferred T cells (1, 2, 3). Recent studies have shown that expansion is dependent on MHC class I and class II molecules, suggesting that the process is driven by recognition of Ag (52, 53, 54). T cell expansion and immune pathology in SCID mice are driven by intestinal bacteria, as transfer of CD45RBhigh CD4+ cells to SCID mice raised under germ-free conditions or under conditions of reduced bacterial flora led to substantially reduced T cell expansion and no colitis (13, 14, 55). Accompanying the dysregulated T cell expansion in mice with colitis was a 5-fold expansion in the number of DC in the MLN. These DC were highly abnormal, as 20–30% expressed CD134L, a molecule present on activated DC, which is present at lower levels on DC present in the MLN (3–8%) or spleen of normal mice. Somewhat paradoxically, despite there being abundant CD134L+ cells in the MLN of mice with colitis, there were few, if any, in the colon. It is possible that in mice with colitis, CD134L+ DC migrate from the colon to the MLN, where they present intestinal Ags to T cells. Such DC migration has been observed after intestinal Ag delivery, and is enhanced by endotoxin and TNF (56, 57).
Although this whole process is dependent on intestinal bacteria, it remains to be established whether they provide peptide Ags or act to enhance the costimulatory capacity of DC that present endogenous Ags, or both. Surprisingly, 9–11% of DC in the MLN of unrestored SCID mice expressed CD134L. Induction of CD134L on DC in vitro has been shown to require CD40 signaling (50). However, the finding that CD134L was expressed on DC in the MLN of unreconstituted SCID mice indicates that in vivo the requirement for T cell-dependent CD40 signaling can be circumvented. Although these activated DC were present in significantly reduced number in SCID mice compared with SCID mice restored with CD45RBhigh CD4+ cells, they may provide the initial costimulatory signals that drive the differentiation of CD45RBhigh cells into pathogenic Th1 cells. Importantly, mice protected from colitis by transfer of Treg cells had similar numbers of CD134L+ DC in the MLN as unrestored SCID mice, suggesting that inhibition of the accumulation of activated CD134L+ DC in the MLN is a feature of the immune-suppressive properties of Treg cells.
Blockade of CD134-CD134L interactions led to amelioration of EAE and of
colitis in a trinitrobenzene sulfonic acid-induced model, in
IL-2-/- mice, and in mice with acute
graft-versus-host disease (30, 40, 19). Although these
results identify this pathway as important in gastrointestinal immune
pathology, they do not identify whether this pathway is involved in T
cell costimulation or migration of T cells into the intestine.
CD134+ cells have been found in the brain in mice
with EAE and in the colon of mice with colitis (30, 40).
In the former case, CD11b+ microglia were shown
to express CD134L, suggesting that CD134L present on APC in
inflammatory lesions may be an important costimulatory molecule driving
proliferation of CD134+ effector cells at sites
of inflammation. Somewhat at odds with these findings, our studies
failed to reveal CD134L expression on cells in the colon of mice with
colitis by flow cytofluorography or in situ by immunohistochemistry.
Furthermore, despite the presence of substantial T cell infiltration,
there were few CD134+ T cells in the inflamed
colon, but abundant expression of CD134 on T cells in the MLN, further
supporting the idea that it is DC expression of CD134L in the MLN that
drives the pathogenic process. However, lack of detectable expression
of CD134 or CD134L in the colon does not rule out the possibility that
this pathway provides T cell costimulation in the colon in addition to
in the MLN, and additional experiments are required to address this. It
has been argued that the inhibition of Th1 inflammatory responses via
blockade of this pathway involves effects on CD134L on the vascular
endothelium and inhibition of migration and not costimulation
(58). Consistent with this, analysis of CD134L expression
using CD134Ig revealed CD134L expression on the inflamed vascular
endothelium in patients with IBD (59). However, our
results are at odds with this hypothesis, as in colitic mice, there was
no detectable CD134L in the colon or on the vascular endothelium.
However, the finding that the frequency of T cells expressing the
gut-homing molecule
47 was significantly
reduced in OX89-treated mice suggests that an impairment in
costimulation can also affect migration as a result of reduced
expression of cell adhesion molecules. Indeed, interaction between
47 and its ligand
mucosal addressin cell adhesion molecule 1 has been shown to be
essential in the development of colitis in the SCID model
(60), and it seems likely that part of the ability of OX89
to inhibit colitis involves reduced expression of this molecule.
Based on these data, we propose a model in which inflammation in the intestine, probably triggered by bacteria, leads to an increase in the recruitment of DC or their precursors from the blood into the lamina propria. In the intestinal environment, these DC sample intestinal Ags (bacterial and/or self) become activated, and migrate to the MLN, where they activate T cells. In the absence of Treg cells, CD134L+ DC drive uncontrolled T cell expansion, which, in the presence of IL-12, leads to the differentiation of Th1 cells. These cells home to the intestine, where, after secondary stimulation, they mediate their effector function. This increases the level of inflammation, which in turn increases DC recruitment to the lamina propria. Based on this model, immune interventions that disrupt this positive feedback loop should inhibit colitis. Consistent with this, in addition to OX89 treatment shown in this study, disruption of IL-12 signaling (7, 11) or of CD154-CD40 interactions (61) also prevented colitis in T cell-restored SCID mice. Normally, Treg cells interrupt this feedback by preventing the accumulation of activated DC in the MLN. Whether this is a result of effects on DC migration or on their activation and survival in the MLN remains to be established.
It is a possibility that dysregulated DC activation and expression of CD134L is a feature of immune pathology that accompanies lymphopenia in a number of models, and that regulation of CD134L expression on DC by Treg cells is one mechanism by which these cells actively control peripheral T cell responses. Based on these studies, targeting of CD134-CD134L interactions may be efficacious for the prevention of a number of T cell-mediated immune pathologies, including IBD and organ-specific autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Current address: Rheumatology Research Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
3 Current address: Tenovus Research Laboratory, University of Southampton, Southampton, U.K.
4 Address correspondence and reprint requests to Dr. Fiona Powrie, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, U.K. E-mail address: fiona.powrie{at}pathology.ox.ac.uk
5 Abbreviations used in this paper: IBD, inflammatory bowel disease; DC, dendritic cell(s); EAE, experimental autoimmune encephalomyelitis; MLN, mesenteric lymph node; Rag, recombinase-activating gene; s, soluble; Treg, regulatory T.
Received for publication October 30, 2000. Accepted for publication March 27, 2001.
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expression by T cells. J. Exp. Med. 187:1225. but not interleukin 4 in the suppression of T helper 1-mediated colitis by CD45RB(low) CD4+ T cells. J. Exp. Med. 183:2669.-chains (CD25). J. Immunol. 155:1151.[Abstract]
7 integrins on mouse lymphocytes: relevance to the mucosal immune system. Eur. J. Immunol. 21:2591.[Medline]
47 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185.[Medline]
1-induced expression of the mucosa-related integrin E on lymphocytes is not associated with mucosa-specific homing. Eur. J. Immunol. 25:1487.[Medline]
47/MAdCAM-1 and OX40/OX40 ligand in the colon and jejunum of patients with inflammatory bowel disease. Gut 45:856.7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells. J. Immunol. 1558:2099.
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|
|
|
|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
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|
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|
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|
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|
|
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|
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|
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|
|
|
|
|
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|
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|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 P C L Beverley Immunology of vaccination Br. Med. Bull., July 1, 2002; 62(1): 15 - 28. [Abstract] [Full Text] [PDF]
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