Blocking inducible co-stimulator in the absence of CD28 impairs Th1 and CD25+ regulatory T cells in murine colitis
Ype P. de Jong1,
Svend T. Rietdijk1,
William A. Faubion1,
Ana C. Abadia-Molina1,
Kareem Clarke1,
Emiko Mizoguchi2,
Jane Tian3,
Tracy Delaney3,
Stephen Manning3,
Jose-Carlos Gutierrez-Ramos3,
Atul K. Bhan2,
Anthony J. Coyle3 and
Cox Terhorst1
1 Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA 2 Immunopathology Unit, Massachusetts General Hospital, Boston, MA 02114, USA 3 Inflammation Division, Millennium Pharmaceuticals Inc., Cambridge, MA 02139, USA
Correspondence to: C. Terhorst; E-mail: terhorst{at}bidmc.harvard.edu
Transmitting editor: W. Strober
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Abstract
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Several autoimmune disease models depend on an imbalance in the activation of aggressor Th1 and CD4+CD25+ regulatory T (Treg) cells. Here we compare the requirement for signals through the co-stimulatory molecules CD28 and inducible co-stimulator (ICOS) in chronic murine colitis, a model for inflammatory bowel disease. We used a colitis model in which disease-causing CD45RBhi T cells alone or in combination with CD4+CD25+ T cells from either CD28-deficient or wild-type donors were transferred into T cell-deficient animals, half of which were treated with ICOS-blocking reagents. Blocking ICOS on the surface of CD28-deficient Th1 cells abrogated development of colitis, whereas blocking CD28 or ICOS alone had little to no effect on disease induction. In contrast to Th1 cells, regulatory T cell functioning depended mostly on CD28 signaling with only a minor contribution for ICOS. We conclude that CD28 and ICOS collaborate to development of murine colitis by aggressor Th1 cells, and that CD28 is required for Treg cells, which should caution against the use of CD28-blocking reagents in inflammatory bowel disease.
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Introduction
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It has become widely accepted that for optimal activation, T cells require co-stimulatory signals in addition to the primary signal through the TCR. If the T cell does not receive co-stimulatory signals, it is rendered anergic or undergoes apoptosis (1).
One such co-stimulatory receptor is CD28, which is constitutively expressed on T cells, and binds to its ligands B7-1 (CD80) and B7-2 (CD86). CD28-mediated co-stimulation plays a critical role in normal T cell activation, as illustrated by in vivo blocking studies in several murine autoimmune disease models. Thus, experimental allergic encephalomyelitis, collagen-induced arthritis and experimental lupus were markedly reduced when the CD28B7 pathway was blocked (24). However, many pathogens have been shown to trigger efficient T cell responses in the absence of CD28 signaling (58), suggesting that other co-stimulatory signals can substitute for the lack of CD28 in infections.
Recently, several new members of the CD28B7 family have been discovered (9,10). Inducible co-stimulator (ICOS), which is up-regulated on T cells upon activation, engages the novel B7rp1 counter-receptor on antigen-presenting cells (APC) (11,12). Stimulation through ICOS was shown to augment IL-2, IL-4, IL-5, IFN-
and tumor necrosis factor production similar to CD28 co-stimulation (11,13). In addition, signals through ICOS were more potent at augmenting IL-10 production than through CD28 co-stimulation (11). Mechanisms to counterbalance the activation signals through CD28 and ICOS are present in the form of two negative regulators of the same gene family, i.e. cytotoxic T lymphocytic antigen (CTLA)-4 and programmed death receptor (PD)-1 (14,15). The indispensable roles of these two negative regulators were best demonstrated by the lymphoproliferative and autoimmune phenotypes respectively of CTLA-4/ and PD-1/ mice (16,17).
Experimental murine colitis (18), a model for human inflammatory bowel disease, differs from many other autoimmune models in that the immune response appears to be directed against bacteria in the lumen of the colon, presented as allo-antigens (1921). One such model, termed the CD45RBhi transfer model, depends on depletion of the CD4+CD25+ regulatory T (Treg) cell (22) subset from CD4+ splenocytes, which induces wasting disease with colitis when transferred into T cell-deficient animals (23,24). This model, like many other experimental autoimmune diseases, is dependent on activated Th1APC interactions (25). Although several co-stimulatory and pro-inflammatory receptors have been implicated in this Th1APC interplay (2630), the variety and abundance of bacterial antigens makes it hard to predict the necessity for CD28 and ICOS co-stimulation in murine colitis models. We therefore tested the need for activating signals through CD28 and ICOS in the induction of experimental colitis by comparing wild-type or CD28-deficient (CD28/) CD45RBhi T cells whilst treating recipient mice with blocking reagents against ICOS. In addition, the need for these co-stimulatory signals on Treg cells was assessed in vivo.
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Methods
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Mice
CD28-deficient (CD28/) mice on the C57Bl/6 (B6) or BALB/c background, B6 wild-type, BALB/c wild-type and B6 recombination-activating gene-1-deficient (RAG/) mice were purchased from Jackson Laboratories (Bar Harbor, ME), BALB/c RAG-2-deficient (RAG/) were from Taconic (Germantown, NY) and F3 backcrossed to BALB/c ICOS/ mice were maintained in a specific pathogen-free facility at Millennium (31). Transfer experiments with CD45RBhi cells only were performed in the B6 background except for mixed 129BALB/c ICOS/ experiments that were performed in the BALB/c background. All experiments in which CD25+ T cells were co-transferred were also performed in the BALB/c background. Experiments were performed along guidelines approved by Beth Israel Deaconess animal committee.
CD45Rbhi and CD4+CD25+ cell transfer
The CD45Rbhi model was generated as described (24,27). From donor spleens, 2 x 105 sorted CD4+ CD45Rbhi cells alone or in combination with 1 x 105 CD4+ CD45Rblo CD25+ cells, all 9599% pure, were resuspended in 400 µl PBS and injected into the tail veins of T cell-deficient RAG/ recipient mice that were between 7 and 10 weeks of age.
Cell preparation and stimulation
Lymphocytes were isolated from the colonic lamina propria (LP) by enzymatic digestion and Percoll (Pharmacia, Peapack, NJ) gradient purification (32). Cells were then stimulated in RPMI with 5% FCS with 1 µg/ml plate-bound anti-CD3
antibody (clone 145-2C11; PharMingen, San Diego, CA), either alone or in combination with 1 µg/ml plate-bound recombinant B7rp1 or B7-1. After 24 h cells were stained for pro-inflammatory receptors. For cytokine production, T cells isolated from the LP were co-cultured with irradiated CHO cells stably transfected with Fc receptors and either B7-1, B7-2, B7rp1 or B7-1 + B7rp1 after addition of 100 ng/ml of anti-CD3
. After 36 h, supernatants were frozen for further analyses.
Cecal antigen was prepared by opening cecums and placing the content in 1 ml of PBS. Bacteria were expelled by mixing with a vortex and residual cecal tissue was removed. After addition of DNase (10 µg/ml), 1 ml of this bacterial suspension was added to 1 ml of glass beads. The cells were disrupted at 5000 r.p.m. in a Mini-Bead Beater (BioSpec Products, Bartlesville, OK) for 3 min and then iced. The glass beads and unlysed cells were removed by centrifuging at 5000 g for 5 min. The lysates were filter sterilized by a 0.2-µm syringe filter (20). For stimulation, adherence-purified and irradiated splenocytes were pulsed overnight with cecal antigen. These were co-cultured with freshly isolated LP lymphocytes for 24 h in the presence of 10 µg/ml of human Ig (Sigma, St Louis, MO), ICOSIg (13) or CTLA-4Ig (Chimerigen, Allston, MA) followed by addition of 0.5 µCi [3H]thymidine (Amersham, Arlington Heights, IL) for 12 h, after which thymidine incorporation was measured.
Antibodies and cytokine detection
ICOSIg and blocking anti-mouse ICOS (clone 12A8) (33) were produced as described, human Ig control and control rat IgG (Jackson ImmunoResearch, West Grove, PA) were purchased. Surface and cytoplasmic staining and flow cytometry analysis were performed as described previously (32), using isotype-matched control antibody staining as the zero value and graphing only lymphocyte and CD4+ gated cells. The following antibodies were used: anti-CD4 (clone RM4-5), CD25 (clone PC61), CD28 (clone 37.15), CD30 (clone mCD30.1), CD45RB (clone 16A), CD154 (clone MR1), CTLA-4 (clone UC10), OX40 (clone OX86) and anti-rat IgG2b (clone RG7/11.1), all from PharMingen, and anti-ICOS (clone12A8). IL-4, IL-10 and IFN-
(PharMingen) in serum and supernatants were detected using standard ELISA kits.
Real-time PCR analysis of RNA levels
After sacrifice, colon pieces were snap-frozen in liquid nitrogen. Total RNA was prepared by a single-step extraction method using RNA STAT-60 according to the manufacturers instructions (Tel-Test, Friendswood, TX). Each RNA preparation was treated with DNase I (Ambion, Austin, TX) at 37°C for 1 h. DNase I treatment was determined to be complete if the sample required at least 38 PCR amplification cycles to reach a threshold level of fluorescence using ß2-microglobulin as an internal amplicon reference. After phenol extraction, cDNA was prepared from the sample using the SuperScript Choice system following the manufacturers instructions (Invitrogen Life Technologies, Carlsbad, CA). Expression profiles ware measured by TaqMan quantitative PCR (Applied Biosystems, Foster City, CA) as described earlier (33).
Disease monitoring and scoring
Mice were weighed twice a week, and monitored for appearance and signs of soft stool and diarrhea. When some mice in an experiment were moribund, which typically occurred between 5 and 7 weeks, all mice in that experiment were sacrificed. They were then scored on a disease activity score that is the sum of four parameters: hunching and wasting were scored 0 or 1, colon thickening 03 and stool consistency 03. Three tissue samples from the proximal, middle and distal colon were prepared for hematoxylin & eosin (H & E) histology. For the histological colitis score, the area most affected was scored on a scale of 03 in each of three criteria: cell infiltration, crypt elongation and the number of crypt abscesses. Histological grades were assigned in a blinded fashion by one pathologist (A. K. B).
Statistical analysis
Weight and cytokine data were analyzed using Prism 3 software (GraphPad, San Diego, CA). P values were calculated using a non-paired t-test. Discrete disease activity and histological scores were analyzed with the MantelHaenszel
2-test using SAS software. Per experiment, individual disease activity and histology scores were calculated as a percentage of the mean of their control group.
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Results
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Both the CD28 and ICOS pathways are up-regulated in mice with colitis
Given the importance of co-stimulatory signals for the activation of T cells in autoimmune diseases, expression of several CD28 family members and their ligands were examined in chronic murine colitis. To this end, LP Th cells from healthy wild-type and colitic RAG/ mice that had received disease-causing wild-type CD45RBhi cells (wild-type CD45RBhi
RAG/) were analyzed for cell-surface expression of CD28, ICOS and CTLA-4. As expected, most CD4+ lymphocytes expressed moderate levels of CD28 which were slightly enhanced in colitis (Fig. 1A), similar to increased expression in human inflammatory diseases (34). In contrast, ICOS was strongly up-regulated on the surface of LP T cells from colitic animals, whereas LP T cells from healthy wild-type mice expressed low amounts of ICOS (Fig. 1A). Interestingly, ICOS expression in colitic animals was similar on wild-type and CD28-deficient (CD28/) T cells, showing that ICOS up-regulation in vivo could occur independent of signals through CD28 (Fig. 1A) (35). No surface expression of CTLA-4 was detectible on CD4+ lymphocytes from healthy control mice, wild-type CD45RBhi
RAG/ mice or CD28/ CD45RBhi
RAG/ mice (data not shown). Intracellular staining for CTLA-4 showed that <3% of CD4+ T cells contained this co-receptor in the LP of either healthy wild-type (data not shown) or colitic mice (Fig. 1A).


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Fig. 1. Differential expression of CD28B7 gene family members in murine colitis. (A) Freshly isolated LP lymphocytes from healthy wild-type mice and colitic mice were double stained with FITC and phycoerythrin-labeled antibodies on their surface (CD28 and ICOS) or intracellularly (CTLA-4). Cells were gated on lymphocyte scatter and CD4 expression; only CD4+ lymphocytes were graphed. (B) Colonic mRNA levels were determined after snap-freezing colons immediately post-mortem. The colon tissue was homogenized with a sonicator using STAT-60 media and RNA was determined by TaqMan RT-PCR (*P < 0.05, **P < 0.01, ***P < 0.001).
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To test whether the ligands that belong to the B7 gene family were expressed in a distinct fashion in the colon of mice with colitis compared to healthy wild-type mice, the amount of mRNA in total colon tissue was measured. Levels of both B7-1 and B7-2 mRNA were almost 7-fold higher in inflamed colons than in healthy wild-type colons (Fig. 1B). In contrast, mRNA for B7rp1, the ligand for ICOS was expressed slightly lower in colitis as compared to healthy colons (Fig. 1B).
These findings show that colon expression of both the CD28 and ICOS pathways is induced by chronic murine colitis, either through up-regulation of ICOS or of the CD28 ligands.
Blocking ICOS in the absence of CD28 protects mice from colitis
Based on their importance in T cell activation and because these pathways were up-regulated in murine colitis, the requirement for signals through CD28 and ICOS in the induction of disease was examined.
First, wild-type CD45RBhi
RAG/ mice were compared to RAG/ mice that had received donor T cells from CD28/ mice (CD28/ CD45RBhi
RAG/). Then, mice treated with either an ICOSIg fusion protein or with anti-ICOS, starting on the day of T cell transfer, were analyzed. Because of a potential additive effect of blocking both the CD28 and ICOS receptors simultaneously, transfer experiments were performed in the Th1-prone B6 strain. This genetic background was more likely to show residual disease while blocking only one co-stimulatory pathway. Surprisingly, the absence of CD28 on the surface of the colitis-inducing T cell subset had only a modest effect on disease outcome. Although CD28/ CD45RBhi
RAG/ mice on average lost 12% less weight than recipients of wild-type donor cells (Fig. 2A), there was only a mild decrease in clinical disease activity score (Fig. 2B) and severity of colitis (Fig. 2C). This was confirmed by histological examination of the colons, where colons from CD28/ CD45RBhi
RAG/ mice displayed slightly less inflammatory cell infiltrate with shorter crypts than colons from wild-type CD45RBhi
RAG/ mice (Fig. 2D and E). Blocking ICOS signaling had even less effect in these experiments. Thus, wild-type CD45RBhi
RAG/ mice treated with ICOSIg started losing weight with the same kinetics as control Ig treated mice (data not shown). At the end of the experiment, they had lost only 7% less weight, and showed similar clinical disease signs and colitis severity as did mice treated with control Ig (Fig. 2AC). This was confirmed by studying donor CD45RBhi T cells from ICOS/ mice that were able to cause disease with similar severity (5.6 ± 0.3) as wild-type donor T cells (5.1 ± 0.1; P = 0.10). In contrast, treating CD28/ CD45RBhi
RAG/ mice with ICOSIg was able to significantly prevent disease compared to control Ig administration (Fig. 2AC). Histological examination showed marked reduction of inflammatory infiltrate and of crypt destruction (Fig. 2F). Similar results were obtained by treating CD28/ CD45RBhi
RAG/ mice (n = 12) with anti-ICOS, which resulted in a 73% decrease in clinical disease severity compared to wild-type CD45RBhi
RAG/ controls (n = 11; P < 0.01).


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Fig. 2. Blocking ICOS on CD28/ T cells attenuates development of colitis. (A) Loss of weight at the end of the experiment as percentage of the initial weight. Recipient mice were transplanted with 2 x 105 wild-type or CD28/ (CD28ko) CD45RBhi T cells and treated with either control Ig (filled circles) or ICOSIg (open circles) twice weekly from the start of the experiment, creating four experimental groups (*P < 0.05, **P < 0.01). (B) Disease activity scores. Clinical appearance, stool and colon thickness were all scored in a blinded fashion on a 03 scale and graphed as a percentage of the wild-type CD45RBhi T cell recipients that received control Ig (*P < 0.05, **P < 0.001). (C) Histology scores. Colons were harvested and fixed in formalin. H & E sections were prepared, and scored in a blinded fashion by one pathologist on a scale of 07 on the basis of crypt elongation, inflammatory infiltrate and crypt abscesses (*P < 0.001). (D) H & E histology of the colon of a representative mouse that had received wild-type donor T cells and that was treated with control Ig throughout the experiment. (E) H & E histology of the colon of a representative mouse that had received CD28/ donor cells and that was treated with control Ig throughout the experiment. (F) H & E histology of the colon of a representative mouse that had received CD28/ donor cells and that was treated with ICOSIg.
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These data show that signals through CD28 and ICOS both contributed to induction of Th1-mediated colitis, but that only blocking them simultaneously could substantially impair the progression of disease.
LP T cells are susceptible to CD28 and ICOS co-stimulation
The lower number of T cells observed in the colons from CD28/ CD45RBhi
RAG/ mice treated with ICOSIg (Fig. 2F) suggested an inhibition of T cell proliferation. To test this, the effect of blocking CD28 or ICOS on antigen-specific T cell proliferation was tested in vitro. As described earlier (20), LP lymphocytes from colitic animals proliferated to cecal antigen upon presentation by professional APC (Fig. 3A). When CD28 was blocked, a complete reduction of antigen-specific proliferation was observed while inhibiting ICOS signaling had no effect on T cell proliferation (Fig. 3A).
To further examine why blocking ICOS inhibited T cell activation in vivo, we measured LP lymphocyte IFN-
production, which is another parameter of T cell activation in colitis. Upon blocking of both ICOS and CD28, a lower IFN-
level was detected in the serum (Fig. 3B). The IFN-
serum values from the other three experimental groups (Fig. 2) were either not affected or minimally affected (Fig. 3B). The effect of co-stimulation through ICOS and CD28 on IFN-
production was also measured in vitro by polyclonal stimulation of freshly isolated LP T cells with low-dose anti-CD3 together with cells expressing B7 receptors. Co-stimulation with B7rp1 more than doubled the amount of IFN-
produced by LP T cells to values higher than B7-1 co-stimulation (Fig. 3C). Combining B7-1 and B7rp1 further augmented IFN-
production, whereas B7-2 co-stimulation had no significant effect (Fig. 3C). Although co-stimulation through ICOS has been shown to augment IL-10 production more than through CD28 (11), no increase in IL-10 could be observed in these in vitro experiments (data not shown).
As CD154 (CD40 ligand/gp39) is involved in Th1macrophage interactions that are critical for the pathogenesis of experimental colitis (27,28) and because ICOS/ mice were shown to have a defect in up-regulating CD154 (36), regulation of CD154 via interference with ICOS was measured both in vivo and in vitro. To test whether signals through ICOS induced CD154 in vivo, mRNA levels in colons from mice in which CD28 and/or ICOS had been blocked were measured. Experimental colitis strongly induced CD154 mRNA levels (Fig. 3D), which correlated with surface staining on LP Th cells (27). CD154 levels were lower in mice treated with anti-ICOS than with control mAb. Furthermore, when CD28/ colitis-inducing T cells were used in conjunction with anti-ICOS, CD154 mRNA levels fell even further, but did not reach values observed in healthy wild-type mice (Fig. 3D). To examine the effect of triggering ICOS on CD154 regulation directly, LP T cells from colitic mice were stimulated with low-dose plate-bound anti-CD3 combined with B7rp1 co-stimulation. Whereas a clear induction of CD154 could be detected when colon T cells were co-stimulated through ICOS (Fig. 3E), no effect on the expression of CD30 (37) or OX40 was observed (data not shown).
These data combined showed that ICOS and CD28 co-stimulation have both similar and distinct effects on LP T cells. Whereas both receptors augment IFN-
production, stimulation through CD28 contributes to proliferation and stimulation through ICOS is able to induce CD154 expression.
Regulatory T cells need CD28 and ICOS to prevent colitis
ICOS signaling is potent at inducing IL-10 production (11), a cytokine necessary for Treg cells to prevent disease (38). Therefore, and because we did not observe any IL-10 augmentation on LP Th1 cells after ICOS co-stimulation, we next set out to investigate whether Treg cells needed signals through either ICOS or CD28 in vivo. To test this, wild-type CD45RBhi T cells were transferred into recipient mice together with wild-type Treg cells. Treatment of recipient mice with ICOSIg or control Ig was initiated at the same time of cell transfer. Interference with signaling through ICOS had no effect on the functioning of wild-type Treg cells. This was apparent by the lack of weight loss in mice that had received wild-type Treg cells, irrespective of whether recipients were treated with ICOSIg (112.9 ± 1.2% of start weight) or control Ig (110.6 ± 2.5%, P = 0.49). Similarly, histological examination of the colon showed that wild-type Treg cells completely prevented disease independent of ICOSIg treatment (Fig. 4B), which was best illustrated by the lack of influx of inflammatory infiltration (Fig. 4C).


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Fig. 4. Treg cells require expression of CD28 rather than expression of ICOS for disease prevention. (A) Histology score of colitis in co-transfer experiments. All mice were transplanted with 2 x 105 wild-type CD45RBhi cells alone (none) or in combination with 1 x 105 Treg cells. At sacrifice, colons were fixed in formalin and prepared for H & E histology. Scores are expressed as a percentage of the mean of mice that received only wild-type CD45RBhi cells (*P < 0.05, **P < 0.01, ***P < 0.001). (B) H & E histology of a colon of a representative mouse that had received wild-type Treg cells. (C) H & E histology of a colon of a representative mouse that had received CD28/ Treg cells and control Ig. (D) H & E histology of a colon of a representative mouse that had received CD28/ Treg cells and ICOSIg.
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A possible effect of blocking ICOS on Treg cells in the absence of CD28 was examined next. For this, Treg cells from CD28/ mice were co-transferred with wild-type CD45RBhi T cells. As expected (39), CD28/ Treg cells were found to be severely impaired in their ability to prevent disease, which was clear from the recurrence of clinical disease (to 58.0 ± 4.4% of control, P < 0.001 compared to wild-type Treg cells) and histological colitis (Fig. 4B and D). Nevertheless, disease in CD28/ Treg cell recipients was not as severe as in mice that had not received any Treg cells (P < 0.001), showing that there was residual suppression by CD28/ Treg cells. Therefore, blocking ICOS signaling on CD28/ Treg cells was studied. This resulted in further impairment of Treg functioning whereby CD28/ Treg recipients treated with ICOSIg lost as much weight as control mice that had not received any Treg cells. They also lost 6.5% more weight loss compared to CD28/ Treg cell recipients treated with control Ig (P < 0.05) and displayed 10% more severe colitis (Fig. 4B). When examined histologically, most mice displayed severe colitis (Fig. 4E), although not quite with the degree of inflammation observed in recipients of CD45RBhi cells alone (Fig. 2D).
These experiments indicated that Treg cells use both ICOS and CD28 signaling to prevent experimental colitis, but that the absence of ICOS can be overcome by CD28-dependent signals.
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Discussion
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Several groups have reported that the ICOS pathway is important for the activation of Th2 cells (13,33,40). Although ICOS co-stimulation could induce both Th1 and Th2 cytokines, its expression was found predominantly on the latter subtype (13). Here we showed that in murine colitis, both ICOS and the two CD28 ligands are up-regulated as compared to healthy wild-type mice. This is in accordance with observations by Totsuka et al., who saw a comparable induction of ICOS on LP mononuclear cells (41). Furthermore, they showed an increase in B7rp1 protein expression in colitic tissue as compared to healthy colon, whilst we observed
30% less B7rp1 mRNA. It has yet to be determined whether lower B7rp1 mRNA levels can still lead to increased protein levels or that other, as of yet undiscovered, ICOS ligands facilitate to the role of ICOS in murine colitis. In the CD45Rbhi colitis model, little effect has been attributed to Th2 cells [(42) and unpublished observations). We therefore prefer the explanation that blocking ICOS, which was expressed on virtually all CD4+ cells in colitis, impaired the activation of Th1 cells in this model. This notion is supported by our observations that Th1 cells were receptive to ICOS co-stimulation in vivo and that no difference in Th2 cytokines was observed in similar experiments reported by Totsuka et al. (41). Furthermore, ICOS blockade has recently been shown to influence disease outcome in two other Th1-dependent disease models, i.e. experimental encephalomyelitis and allograft rejection (31,43). However, in contrast to these models and to the above-mentioned report in murine colitis (41), no effect was observed by blocking ICOS alone in our experiments. As suggested by Totsuka et al., the ameliorating effects of anti-ICOS on established colitis that they observed may be explained by the apoptosis-inducing properties of their antibody. Because in both our and their experiments no effect was observed when CD45RBhi T cells from ICOS/ mice were compared to cells from wild-type mice, we conclude that ICOS signaling is dispensable for the development of murine colitis (41). Similarly, only a very mild amelioration was seen when CD28/ T cells were used. This could be explained by the strong antigenic stimulation in colitis, which influences the requirements for co-stimulatory signals (6), as well as by the observations that many pathogens bypass the necessity for CD28 co-stimulation (58). We therefore hypothesize that certain as yet unidentified pathogens in experimental colitis provide such strong antigenic stimulation that only blocking ICOS in combination with CD28 can impair Th1 cells from causing disease.
One mechanism by which ICOS contributes to activation of T cells could be its ability to activate the CD154CD40 pathway (11,36), which was shown to be important in several colitis models (27,28,44). We indeed demonstrated that signal transduction through ICOS could up-regulate CD154 on LP T cells in vitro. In addition, decreased CD154 mRNA expression in LP T cells was found after in vivo blocking of ICOS. Because blocking of ICOS by itself did not influence disease outcome, alternative, CD28-dependent pro-inflammatory pathways must be in place to compensate for lack of ICOS-induced CD154 expression, e.g. ICAM-1, 4-1BB or LIGHT (45,46).
Compared to wild-type Treg cells, Treg cells from CD28/ mice were markedly impaired in their ability to control colitis. A recent report indicates that T cells isolated from CD28/ mice were able to prevent colitis (47). The ratio of CD28/ Treg cells to colitis-inducing T cells that were co-transferred was almost similar in that and our study, 1:1 and 1:2 respectively. Because wild-type Treg cells were able to prevent colitis when transferred in a 1:8 ratio (24), the difference in cell number cannot explain the distinct outcomes. Our results suggest that under physiological conditions, the absence of CD28 on Treg cells would impair their control over pathogenic cells. In contrast, no effect was observed by blocking ICOS alone in our Treg cell co-transfer experiments. This was surprising for three reasons. First, ICOS was more potent than CD28 at inducing IL-10, a cytokine necessary for Treg cell functioning (38). This was recently confirmed in vivo where blocking ICOS could inhibit the IL-10-mediated protection of Treg cells in a Th2-driven allergic asthma model (48). Nevertheless, we found that its role in vivo only became apparent after CD28 had been blocked. This demonstrated that CD28-dependent signals are sufficient for IL-10 production by Treg cells that can inhibit our Th1-driven disease, with a requirement for ICOS only in the absence of CD28 signaling. Second, Treg cells need CD154 expression to function optimally (49). This could only be explained by the hypothesis that Treg cells, in contrast to Th1 cells, have no requirement for ICOS in the induction of CD154. Finally, induction of experimental allergic encephalomyelitis was exacerbated in the absence of ICOS, which was suggested to be due to impaired IL-13 production (43,50). Although impaired Treg cell activation was not explicitly demonstrated in those studies, further studies on the requirement of Treg cells for IL-13 may elucidate these findings.
Interfering in the B7CD28 pathway is a likely strategy to modulate autoimmune diseases and protect allogeneic grafts (51). However, our data show that in the case of experimental colitis, blocking CD28 may result in the exacerbation of disease by impairing protective Treg cells more than aggressor Th1 cells. As noted, a similar observation was published by Salomon et al. who showed that breeding the NOD mouse strain to a B7-1/2-deficient background resulted in accelerated onset of spontaneous diabetes (39). This and our observation suggest caution should be exercised in the use of B7CD28 blocking reagents in human inflammatory bowel disease until it has been established how CD28 can be blocked specifically on Th1 cells. Furthermore, in contrast to the apoptosis-inducing anti-ICOS antibody used by Totsuka et al., our experiments indicate that little effect may be expected by blocking CD28 or ICOS alone and that only double blockade would improve the outcome of colitis.
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Acknowledgements
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We thank Drs D. Podolsky and N. Fischer for critically reviewing the manuscript, and J. Daley for expert cell sorting. Supported by grants from the National Institutes of Health (DK52510 to C. T.; DK47677 to A. K. B.; DK43351 to C. T. and A. K. B.), and a research fellowship from the Crohns and Colitis Foundation of America (Y. P. J.).
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Abbreviations
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APCantigen-presenting cell
CTLAcytotoxic T lymphocytic antigen
H & Ehematoxylin & eosin
ICOSinducible co-stimulator
LPlamina propria
PDprogrammed death receptor
RAG-/-recombination-activating gene-deficient mice
TregCD4+CD25+ regulatory T cell
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