The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Asseman, C.
Right arrow Articles by Powrie, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asseman, C.
Right arrow Articles by Powrie, F.
The Journal of Immunology, 2003, 171: 971-978.
Copyright © 2003 by The American Association of Immunologists

Colitogenic Th1 Cells Are Present in the Antigen-Experienced T Cell Pool in Normal Mice: Control by CD4+ Regulatory T Cells and IL-101

Chrystelle Asseman*, Simon Read{dagger} and Fiona Powrie2,{dagger}

* La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and {dagger} Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ regulatory T cells have been shown to prevent intestinal inflammation; however, it is not known whether they act to prevent the priming of colitogenic T cells or actively control these cells as part of the memory T cell pool. In this study, we describe the presence of colitogenic Th1 cells within the CD4+CD45RBlow population. These pathogenic cells enrich within the CD25- subset and are not recent thymic emigrants. CD4+CD45RBlow cells from germfree mice were significantly reduced in their ability to transfer colitis to immune deficient recipients, suggesting the presence of commensal bacteria in the donor mice drives colitogenic T cells into the Ag-experienced/memory T cell pool. This potentially pathogenic population of Ag-experienced T cells is subject to T cell-mediated regulation in vivo by both CD4+CD25+ and CD4+CD25- cells in an IL-10-dependent manner. Furthermore, administration of an anti-IL-10R mAb to unmanipulated adult mice was sufficient to induce the development of colitis. Taken together, these data indicate that colitogenic Th1 cells enter into the Ag-experienced pool in normal mice, but that their function is controlled by regulatory T cells and IL-10. Interestingly, IL-10 was not absolutely required for CD4+CD25+ T cell-mediated inhibition of colitis induced by transfer of naive CD4+CD45RBhigh cells, suggesting a differential requirement for IL-10 in the regulation of naive and Ag-experienced T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is now appreciated that disruption of immune regulatory networks, including deletion of cytokine genes, cell signaling molecules, and alterations in T cell subsets, can lead to an inflammatory bowel disease (IBD3)-like syndrome in mice (1, 2). In many of the IBD models, intestinal inflammation is the consequence of the development of a dysregulated Th1 response driven by resident bacteria (3, 4, 5, 6, 7). This is relevant to IBD in humans, as Th1 responses are elevated in the intestine of patients with Crohn’s disease (8, 9). Furthermore, PBLs from patients with IBD proliferate in response to intestinal bacteria, whereas those from normal individuals do not (10), suggesting that IBD may develop as a consequence of a loss of tolerance to enteric bacteria.

Previous studies from this laboratory and others have shown that transfer of CD4+CD45RBhigh T cells, a predominantly naive population, from the periphery of normal mice to SCID recipients leads to the development of a Th1-mediated colitis with similarities to IBD in humans (3, 11, 12, 13, 14, 15). Intestinal inflammation is driven by resident bacteria, as colitis does not develop after T cell transfer to immune deficient recipients raised under germfree (GF) or restricted flora conditions (16, 17). T cells capable of responding to enteric bacteria are present in T cell-restored immune deficient mice with colitis, and are most likely involved in the pathogenesis of the disease (7).

Colitis induced by transfer of CD4+CD45RBhigh T cells can be prevented by cotransfer of cells contained within the Ag-experienced CD4+CD45RBlow population (11, 12). Regulatory T (TR) cell function is enriched within the 30% of CD4+CD45RBlow cells that express CD25 (18), the same population that has been found to suppress a number of T cell-mediated responses, including autoimmune disease (19, 20), allograft rejection (21), antitumor immunity (22), and T cell activation in vitro (23, 24). However, control of immune pathology is not restricted to CD4+CD25+ cells, and there is evidence that CD4+CD25- T cells also possess some regulatory activity (18, 20, 25), although the relationship between these phenotypically distinct subsets of TR cells is not known.

The mechanisms by which TR cells regulate immune responses remain controversial (26, 27). Administration of anti-TGF-{beta} (28) or anti-IL-10R mAb (29) abrogates the ability of CD4+CD45RBlow cells to inhibit colitis, suggesting these cytokines are involved in the mechanism of immune suppression. TGF-{beta} is also required for suppression of intestinal inflammation by CD4+CD25+ cells (18); however, it is not known whether the same is true of IL-10. Indeed, there is conflicting data on the requirement for IL-10 in suppression mediated by CD4+CD25+ cells. Although CD4+CD25+ cells from IL-10-/- mice fail to control the proliferation of the progeny of CD4+CD45RBhigh cells in vivo (25), inhibition of autoimmune gastritis (30) and T cell activation in vitro occurs independently of IL-10 (23, 24).

Although it is clear that thymocytes (31) and naive T cells can induce colitis, the colitogenic potential of cells within the Ag-experienced CD45RBlow pool is less well characterized. In general, transfer of CD4+CD45RBlow T cells does not induce colitis (11, 12, 13, 16, 32), although exceptions to this have been observed (14). In the latter case, it remains to be determined whether the colitis reflects the activity of naive cells, recent thymic emigrants (RTE), or Th1 effector/memory cells. The answer to this has important implications for our understanding of how TR cells regulate T cell responses in vivo, as the presence of primed colitogenic Th1 cells in the CD45RBlow population would suggest that TR cells do not control T cell responses at the level of activation. In this study, we have further investigated the ontogeny of colitogenic T cells within the CD4+CD45RBlow population. Our data suggest that these cells are not RTE, but represent Ag-experienced Th1 cells that are driven by resident bacteria. The pathogenic potential of these cells can be controlled by CD4+ TR cells and IL-10.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free BALB/c, BALB/c IL-10-deficient (IL-10-/-), C.B-17 SCID mice, 129/SvEv, 129/SvEv recombination-activating gene (RAG)-2-deficient (RAG-2-/-), and 129/SvEv IL-10-/- mice were bred under specific pathogen-free (SPF) conditions and maintained in microisolator cages with filtered air at the Biomedical Services Unit at John Radcliffe Hospital (Oxford, U.K.). All colonies used in these experiments were free of Helicobactor hepaticus, as assessed by PCR (33). GF BALB/c mice were obtained from the Center de Développement des Techniques Avanées pour L’Experimentation Animale (Orléans, France). Mice were used at 8–12 wk of age, unless specified. For thymectomy, 6-wk-old BALB/c mice were anesthetized with Hypnorm (Janssen Pharmaceuticals, Piscataway, NJ) and Hypnoval (Roche, Nutley, NJ) before removal of both lobes (34). All procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986.

Antibodies

The following Abs were used for cell purification: YTS169 (35), 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 (36); FITC-conjugated anti-mouse CD45RB (clone 16A; BD PharMingen, Oxford, U.K.); CyChrome-conjugated anti-mouse CD4 (clone RM4-5; BD PharMingen); biotinylated anti-mouse CD25 (clone 7D4; BD PharMingen); and PE-conjugated streptavidin (BD PharMingen). PerCP-conjugated anti-mouse CD4 (clone RM4-5; BD PharMingen) and PE-conjugated anti-mouse TCR {beta}-chain (clone H57-597; BD PharMingen) were used for analysis of T cell populations in reconstituted SCID mice. FITC-conjugated anti-mouse IFN-{gamma} (clone XMG1-2; BD PharMingen) and PE-conjugated anti-mouse TNF-{alpha} (clone XT-22; BD PharMingen) were used for intracellular cytokine staining. For in vivo use, anti-mouse IL-10R mAb (clone 1B1.2) (37), and GL113, an isotype control mAb (rat IgG1), were purified from hybridoma supernatant by affinity chromatography and shown to contain <0.1 EU endotoxin per mg of protein.

Purification of T cell subsets

CD4+ T cell subsets were isolated from spleen or mesenteric lymph nodes (MLN), as described previously (12). 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 Biotech, Wirral, U.K.). The resulting CD4+-enriched population was stained with CyChrome-conjugated anti-CD4, FITC-conjugated anti-CD45RB, and for some experiments biotinylated anti-CD25 mAb and streptavidin-PE. Subpopulations of CD4+ cells were isolated by cell sorting on a FACSVantage (BD Biosciences, Oxford, U.K.). All populations were >98.5% pure on reanalysis. In some experiments, mice were thymectomized 6–8 wk before isolation of CD4+ T cells.

T cell reconstitution and Ab treatment

C.B-17 scid mice or RAG-2-/- mice were injected i.p. with sorted CD4+ T cell subpopulations in PBS. mAbs were also injected i.p. in PBS the day after T cell reconstitution, and hence once per week for the duration of the experiment.

Histological examination

Colons were removed from mice 6–8 wk after T cell reconstitution and fixed in buffered 10% Formalin. Paraffin-embedded sections (6 µm) were cut and stained with H&E. Inflammation was scored in a blinded fashion, on a scale of 0–4, representing no changes to severe inflammation. 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 mild to moderate epithelial hyperplasia and mild to moderate mucin depletion from goblet cells; grade 3, moderate inflammatory cell infiltrates that were sometimes transmural, with moderate to severe epithelial hyperplasia and mucin depletion; grade 4, marked inflammatory cell infiltrates that were often transmural and associated with crypt abscesses and occasional ulceration, with marked epithelial hyperplasia, mucin depletion, and loss of intestinal glands.

Cell preparation and cytokine analysis

Lamina propria (LP) lymphocytes were purified, as described (3). Briefly, colons were cut into 0.5- to 1.0-cm pieces and incubated in Ca- and Mg-free PBS containing 10% heat-inactivated FCS (Invitrogen, Paisley, U.K.) and 5.0 mM EDTA to remove the epithelial layer. The remaining tissue was further digested with collagenase/dispase (100 U/ml; Sigma-Aldrich, Poole, U.K.), and the LP cells were then layered on a Percoll gradient (Amersham Pharmacia Biotech, Amersham, U.K.). The lymphocyte-enriched population was recovered after centrifugation (600 x g, 20 min) at the 40–75% interface. The resulting population was labeled with fluorochrome-conjugated Abs recognizing CD4 and TCR {beta}-chain, and the proportion of CD4+ T cells was determined by flow cytometric analysis on a FACSort using CellQuest software (BD Biosciences). For detection of cytokines, freshly isolated LP lymphocytes were cultured for 12 h in RPMI 1640 (Invitrogen) containing 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, and 100 U/ml each of penicillin and streptomycin in 24-well flat-bottom plates coated with anti-mouse CD3{epsilon} (10 µg/ml) (clone 145-2C11, CRL1975; ATCC). Brefeldin A (10 µg/ml; Sigma-Aldrich) was added for the final 2 h of incubation, and surface and cytoplasmic staining was performed, as described previously (38). Labeled cells were analyzed on a FACSort using CellQuest software.

Statistical analysis

Where indicated, colitis scores were compared using a Mann-Whitney U test, and differences were considered statistically significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-10 controls pathogenic T cells contained within the CD4+CD45RBlow population

The essential role that IL-10 plays in intestinal homeostasis is illustrated by the fact that IL-10-/- mice develop an IBD-like syndrome (39, 40) and mount a pathogenic Th1 response toward the murine intestinal pathogen H. hepaticus (41). Previously, we found that CD4+CD45RBlow T cells from IL-10-/- mice, but not wild-type (wt) mice, were able to induce colitis after transfer into immune deficient recipients (29). This demonstrates that pathogenic T cells are present in the Ag-experienced pool of IL-10-/- mice; however, it is not clear whether this occurs as a consequence of the lack of IL-10 through ontogeny, or whether colitogenic T cells are present in the Ag-experienced pool of normal mice, but remain silent as a result of active control by IL-10.

To investigate this, CD4+CD45RBlow cells were isolated from the spleen or MLN of wt mice and injected into SCID mice together with anti-IL-10R mAb or an isotype-matched control mAb. As expected, colons from mice restored with CD4+CD45RBlow cells isolated from either spleen or MLN and treated with isotype control mAb exhibited no detectable pathological changes (Figs. 1 and 2A). In contrast, 80–90% of CD4+CD45RBlow cell-restored mice treated with anti-IL-10R mAb showed characteristic features of intestinal inflammation, including marked epithelial cell hyperplasia, leukocytic infiltration of the mucosa, and depletion of goblet cells (Fig. 2B). Disease induced by transfer of CD4+CD45RBlow cells together with anti-IL-10R treatment had similar characteristics to that induced by transfer of CD4+CD45RBhigh cells (Fig. 2C), including the presence of high numbers of IFN-{gamma}- and TNF-{alpha}-expressing CD4+ Th1 cells in the large intestine (Table I) (29). Importantly, induction of colitis with anti-IL-10R mAb was strictly dependent on the presence of CD4+ T cells, as unreconstituted SCID mice treated with anti-IL-10R failed to show any signs of inflammation (data not shown). Taken together, these data indicate that potentially pathogenic cells are contained within the CD4+CD45RBlow subset, but that their activity is controlled by IL-10.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Induction of colitis in mice restored with wt CD4+CD45RBlow T cells and treated with anti-IL-10R mAb. C.B-17 SCID mice were reconstituted with 2 x 105 CD4+CD45RBlow cells isolated from spleen or MLN and treated with Abs, as indicated (1 mg the day after T cell reconstitution and 0.5 mg/wk thereafter). Eight to twelve weeks after reconstitution, mice were killed and colonic pathology was assessed. Data from four independent experiments. Whether SCID mice received CD4+CD45RBlow cells isolated from spleen or MLN, administration of anti-IL-10R mAb resulted in significant disease: p < 0.001.

 


View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 2. Representative photomicrographs of the colons of C.B-17 SCID mice after transfer of CD4+ T cells. The colons in mice restored with wt CD4+CD45RBlow cells and treated with isotype control mAb (A) were histologically normal; however, mice receiving CD4+CD45RBlow cells in combination with anti-IL-10R mAb developed severe colitis (B). Injection of 4 x 105 CD4+CD45RBhigh cells also resulted in the development of severe colitis (C); this was prevented by cotransfer of wt CD4+CD25+ T cells (not shown). Inhibition of colitis mediated by CD4+CD25+ cells was not abrogated by administration of anti-IL-10R mAb (D). In addition, IL-10-/- CD4+CD25+ cells also retained the ability to inhibit the development of colitis induced by transfer of CD4+CD45RBhigh cells (E). Original magnification: x250.

 

View this table:
[in this window]
[in a new window]
  		Table I. Expansion of Thl cells in the LP of mice with colitis induced by anti-IL-10R treatment of SCID recipients restored with wt CD4+ CD45RBlow T cellsa

 
Pathogenic cells within the CD45RBlow subset are not RTE and are driven into the Ag-experienced pool by commensal bacteria

The CD4+CD45RBlow T cell population is heterogeneous, containing TR cells (11, 12), Ag-experienced/memory cells (42), and, in the rat, RTE (43). The latter were found to be responsible for the diabetogenic potential associated with CD4+CD45RClowCD25- cells (20). To determine whether the pathogenicity revealed by anti-IL-10R mAb treatment was the result of the expansion of unprimed RTE, the CD45RBlow population was isolated from mice thymectomized 5–8 wk previously. Because postthymic maturation of RTE is thought to occur within 1–2 wk, these mice can be considered to be devoid of RTE (44, 45). As shown in Fig. 3, CD4+CD45RBlow cells isolated from these mice, although devoid of pathogenicity themselves, were able to induce colitis in the presence of anti-IL-10R mAb. These results suggest that the colitogenic T cells within the CD4+CD45RBlow population are mature peripheral T cells and do not represent the activity of RTE.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. Pathogenic T cells in the CD4+CD45RBlow pool are not RTE, but are driven by exposure to bacteria in the donor. C.B-17 SCID mice were reconstituted with 2 x 105 splenic or MLN CD4+CD45RBlow cells from donor mice, as indicated, and treated with Abs, as indicated in Fig. 1. For some experiments, donor mice were thymectomized at least 8 wk before sacrifice; the absence of thymic tissue was confirmed on sacrifice. For other experiments, donor mice were taken from colonies housed under either GF or SPF conditions. Eight weeks after reconstitution, SCID mice were killed and colonic pathology was assessed. Data from four independent experiments. In the presence of anti-IL-10R mAb, CD4+CD45RBlow cells from SPF mice induce significantly more severe colitis compared with the equivalent population from GF mice (p < 0.01).

 
Development of colitis after transfer of CD4+CD45RBhigh cells is dependent on the resident flora (16, 17). However, this population isolated from GF mice is still able to induce colitis (unpublished observations), suggesting naive T cells differentiate into colitogenic Th1 cells upon exposure to resident bacteria in the immune deficient recipient. To investigate whether the presence of colitogenic T cells within the CD4+CD45RBlow population requires prior exposure to enteric bacteria, this population was isolated from the spleens of GF mice and tested for its ability to induce colitis when transferred to immune deficient recipients treated with anti-IL-10R mAb. As shown in Fig. 3, the incidence and severity of colitis were significantly reduced when CD4+CD45RBlow cells were taken from GF mice, suggesting that the pathogenic T cells in the CD4+CD45RBlow population represent a primed population of T cells driven by resident bacteria.

Pathogenic cells are enriched within the CD4+CD45RBlowCD25- subset

To determine whether the colitogenic T cells within the Ag-experienced pool reside together with TR cells within the naturally activated CD25+ subset or are present within the reciprocal CD25- subset, CD4+CD45RBlow cells were fractionated into CD25+ and CD25- subsets and transferred to SCID mice. CD4+CD45RBlowCD25- cells were able to expand in immune deficient recipients and migrate to the LP. However, this population, isolated from either spleen or MLN, did not induce inflammation unless the recipients were treated with anti-IL-10R mAb (Table II). Consistent with this, CD4+CD45RBlowCD25- cells from IL-10-/- mice also accumulated in the colon and were as capable of inducing colitis as wt CD4+CD45RBlowCD25- cells transferred to recipients treated with anti-IL-10R (Table II).


View this table:
[in this window]
[in a new window]
  		Table II. In the absence of IL-10, pathogenic T cells are revealed in both CD25+ and CD25- subsets of CD4+CD45RBlow T cells

 
In contrast, wt CD4+CD45RBlowCD25+ T cells isolated from the spleen or MLN expanded poorly in SCID recipients. In line with a previous report (25), ~10-fold fewer cells were found to accumulate in the colon than after transfer of wt CD4+CD45RBlowCD25- cells (8.0 x 103 ± 0.4 x 103 compared with 1.1 x 105 ± 0.7 x 105 LP CD4+), and in the absence of anti-IL-10R mAb, these populations did not induce colitis (Table II). Strikingly, treatment with anti-IL-10R led to development of colitis only after transfer of CD25+ cells isolated from MLN and not from spleen (Table II). A similar frequency of mice developed colitis (6 of 17; Table II) after transfer of CD4+CD45RBlowCD25+ cells from the MLN of IL-10-/- mice, consistent with the observations of others (25). Taken together, these data indicate that Ag-experienced, potentially pathogenic T cells are enriched within the CD4+CD45RBlowCD25- subset present in both the spleen and MLN, and that their ability to cause disease is regulated by the presence of IL-10. In addition, in the absence of IL-10, pathogenic T cells can also be revealed within the CD25+ population taken from MLN, but not from the spleen. The difference between MLN and spleen may reflect a higher frequency of chronically stimulated bacteria-reactive T cells in the former, and is consistent with the finding that the presence of colitogenic T cells in the CD45RBlow pool is dependent on exposure to bacteria in the donor.

Treatment with anti-IL-10R induces colitis in normal mice

It is possible that development of colitis in immune deficient mice restored with CD4+CD45RBlow cells and treated with anti-IL-10R is a consequence of expansion of pathogenic cells in a lymphopenic environment, and that colitis in IL-10-/- mice is attributable to a lack of IL-10 throughout ontogeny. However, treatment of normal BALB/c mice with anti-IL-10R led to the induction of severe colitis in 8 of 12 treated mice (Fig. 4; mean colitis score, 3.8 ± 0.0; n = 8), demonstrating that IL-10 is actively required in adult mice to maintain intestinal homeostasis.



View larger version (107K):
[in this window]
[in a new window]
  		FIGURE 4. Representative photomicrographs of the colons of wt BALB/c mice after administration of anti-IL-10R mAb. Wt BALB/c mice were treated with anti-IL-10R mAb (B) or isotype control Abs (A), 0.5 mg/wk from 5 wk of age for 8 wk. Mice were sacrificed 7 days after the final injection of Ab. Eight of twelve mice treated with anti-IL-10R developed severe colitis. Original magnification: x250.

 
IL-10 is required for the control of colitis induced by Ag-experienced, but not naive T cells

Transfer of wt CD4+CD25+ TR cells prevents the development of colitis induced by cells contained within the IL-10-/- CD45RBlowCD25- population (2 x 105 IL-10-/- CD45RBlowCD25- cells, 6 of 7 colitic; 2 x 105 IL-10-/- CD45RBlowCD25- + 1 x 105 CD25+ cells, 1 of 7 colitic), indicating that in addition to regulating naive T cells, CD25+ TR cells also suppress Ag-experienced colitogenic T cells. This regulation is IL-10 dependent, as anti-IL-10R treatment induced colitis in recipients of unseparated CD45RBlow cells (Fig. 1), which are a mixture of approximately one-third CD25+ and two-third CD25- cells. The requirement for IL-10 in the suppression of colitis induced by transfer of naive CD4+CD45RBhigh cells has only been examined using unseparated CD45RBlow cells as a source of TR cells (29). Given that anti-IL-10R treatment reveals pathogenic T cells within the CD45RBlow pool, it was important to determine whether the regulation of naive T cells by CD4+CD25+ TR cells also requires IL-10.

To this end, CD4+CD45RBhigh cells were transferred together with splenic CD4+CD25+ cells into recipient mice treated withisotype control or anti-IL-10R mAb. Splenic CD4+CD25+ cells were used because in contrast to the equivalent population taken from the MLN, anti-IL-10R treatment did not reveal the presence of pathogenic cells. Cotransfer of CD4+CD25+ cells significantly inhibited the development of colitis induced by CD4+CD45RBhigh cells, even in the presence of anti-IL-10R (Figs. 2D and 5). However, IL-10 does appear to play some role in CD4+CD25+ TR cell-mediated regulation of naive T cells, as administration of anti-IL-10R led to a small, but significant increase in the incidence of colitis compared with isotype control-treated mice (p < 0.02). Importantly, CD4+CD25+ cells themselves do not have to synthesize IL-10, as this population isolated from IL-10-/- mice was able to inhibit colitis induced by wt CD4+CD45RBhigh cells (Figs. 2E and 5).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 5. Control of colitogenic T cells within the naive T cell pool by CD4+CD25+ T cells is IL-10 independent. C.B-17 scid mice received 4 x 105 CD4+CD45RBhigh cells alone or in combination with 1 x 105 CD4+CD25+ T cells from spleen of wt BALB/c or IL-10-/- mice. mAb treatments were as described in Fig. 1. Eight weeks posttransfer, mice were sacrificed and tissues were taken for histological examination. Data from three independent experiments. In the presence of anti-IL-10R mAb, wt CD4+CD25+ cells confer significant inhibition of colitis (p < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is now well established that TR cells can control a number of T cell-mediated immune diseases (26, 46, 47). Whether TR cells act to prevent the priming of potentially pathogenic T cells or actively control these cells as part of the Ag-experienced/memory T cell pool is not known. In this study, we extend previous findings to show that colitogenic T cells reside alongside TR cells in the Ag-experienced T cell pool in normal mice. These cells are not RTE and are driven into the Ag-experienced T cell pool by exposure to commensal bacteria in the donor. The pathogenic potential of these cells is controlled by both CD4+CD45RBlowCD25+ and CD4+CD45RBlowCD25- TR populations and is highly dependent on IL-10. Surprisingly, control of colitogenic T cells within the naive CD4+CD45RBhigh T cell population by CD4+CD25+ TR cells is less dependent on IL-10, suggesting that TR populations may use distinct mechanisms to inhibit intestinal inflammation depending on the nature of the immune response that drives the inflammation.

The CD4+CD45RBlow T cell pool is heterogeneous, containing Ag-experienced cells (42, 48), TR cells (49, 50), and RTE. In rats, RTE reside within the CD4+CD45RClow population (43), and removal of these cells prevents transfer of diabetes by CD4+CD45RClowCD25- cells, suggesting RTE can contain pathogenic autoreactive cells (20). It has been reported that transfer of CD4+CD45RBlow cells into immune deficient recipients can induce colitis (14); however, it was not established whether the colitogenic T cells derive from naive RTE or Ag-experienced cells. In this study, we show that the colitogenic cells within CD4+CD45RBlow T cell pool are enriched within the CD25- fraction and are present in both spleen and MLN. These cells are not RTE, and their activity is significantly reduced in the CD45RBlow population isolated from GF mice, suggesting that commensal bacteria in the host drive these pathogenic T cells into the Ag-experienced pool. The most straightforward interpretation of these data is that bacteria-reactive Th1 cells are primed in donor mice, but fail to induce pathology as a result of their control by IL-10.

It is notable that in the presence of the anti-IL-10R mAb, CD4+CD45RBlowCD25+ cells taken from the MLN, but not the spleen, were also able to induce colitis. Whether this is a result of differential homing properties or reflects an increased frequency of recently activated bacteria-reactive T cells in the MLN as compared with the spleen is not known. It does, however, emphasize the limitations of using expression of CD25 as a marker unique to CD4+ TR cells, and illustrates that the make-up of cells within the CD25+ population varies with anatomical location.

The finding that anti-IL-10R mAb treatment reveals colitogenic Th1 cells that enrich within the CD4+CD45RBlowCD25- population emphasizes the importance of IL-10 in the control of Ag-experienced T cell responses. This is not an artifact of T cell transfer to immune deficient recipients, as treatment of adult BALB/c mice with anti-IL-10R mAb was sufficient to induce colitis with similar kinetics and severity to that induced by T cell transfer. Whether IL-10 is produced by T cells or by host-derived cells such as myeloid cells is not clear. However, the observation that IL-10-/- CD4+CD45RBlowCD25- cells are able to induce pathology when transferred to IL-10+/+ recipients would suggest that in wt mice, T cells present within the CD25- population regulate the pathogenic cells that coexist within this population via the production of IL-10. CD4+CD25- cells have also been shown to inhibit colitis induced by transfer of naive T cells, albeit with reduced potency when compared with CD4+CD25+ TR cells (18), and to inhibit both diabetes (20) and experimental autoimmune encephalomyelitis (51). The data presented in this study suggest that IL-10 is an important molecule for suppression mediated by these cells; indeed, among CD4+ T cells, IL-10 production enriches within the CD4+CD45RBlowCD25- population (52) (our own unpublished results). Consistent with this, it was recently reported that infection of wt mice with H. hepaticus results in the generation of H. hepaticus-specific TR cells. The regulatory activity was found to enrich within the CD4+CD45RBlowCD25- population and to be mediated via IL-10 (53). It is possible that the IL-10-dependent TR cells within the CD45RBlowCD25- population represent the in vivo counterpart of the in vitro derived T regulatory type 1 cells shown to inhibit both colitis (54) and experimental autoimmune encephalomyelitis (55).

An area of controversy within the TR cell field is how CD4+CD25+ TR cells function to inhibit T cell-mediated immune pathology (26, 46). Our data concerning control of intestinal inflammation and the role of IL-10 suggest that the situation is complex and that multiple mechanisms of suppression may operate in vivo. Thus, while control of Ag-experienced colitogenic T cells was found to be highly dependent on IL-10, suppression of naive T cell responses was less dependent, as treatment with anti-IL-10R induced only a marginal reduction in the ability of CD4+CD25+ cells to prevent colitis. Furthermore, CD4+CD25+ cells isolated from IL-10-/- mice retained the ability to inhibit colitis induced by CD4+CD45RBhigh cells, indicating that IL-10 secretion by CD4+CD25+ TR cells themselves is not required. Depletion of CD25+ cells from IL-10-/- CD4+ T cells before transfer to RAG-2-/- mice led to an enhanced incidence of colitis (30), providing support for the presence of CD4+CD25+ T cell-mediated IL-10-independent mechanisms of intestinal regulation. TGF-{beta} has been shown to be required for CD4+CD25+ TR cell-mediated suppression of colitis induced by CD4+CD45RBhigh cells (18), suggesting that this cytokine may be sufficient to prevent colitis induced by the activation of primarily naive cells, whereas IL-10 is required to control previously activated/differentiated Th1 cells. CD4+CD25+ cells have been shown to express membrane-bound TGF-{beta} (52, 56), suggesting TR themselves may be a source of this cytokine. A recent report has shown that activated/memory cells become refractory to TGF-{beta} signaling via down-regulation of TGF-{beta} receptor type II expression (57). This could be reversed by IL-10, indicating that IL-10 may play an important role in potentiating the effects of TGF-{beta} on differentiated effector T cells.

It should be noted that in some studies, IL-10 synthesis by CD4+CD25+ cells has been found to be required for prevention of CD4+CD45RBhigh cell-induced immune pathology in immune deficient recipients (25, 58). This suggests that the state of differentiation of the pathogenic T cells is not the only factor that influences mechanisms of regulation. As intestinal inflammation was not assessed in these studies, it is difficult to make direct comparisons between these studies and our own; however, differences may be attributable to the use of immune deficient recipients of different genetic background or to the environmental factors that drive the inflammatory response. The nature of the endogenous microbiota in immune deficient recipients may be an important and variable factor when comparing results between different laboratories. Recently, we have found that control of both T cell-dependent and T cell-independent intestinal inflammation induced by the mouse pathogen H. hepaticus requires IL-10 secretion by CD4+CD25+ cells (59). Thus, in the presence of a potent proinflammatory stimulus, such as H. hepaticus, IL-10 is required in addition to TGF-{beta} to control naive T cells. Although TGF-{beta} may act directly on T cells (60, 61, 62), IL-10 may serve to inhibit proinflammatory cytokine production, particularly IL-12, by macrophages and DC (63, 64, 65). In support of this, IL-10 was shown to enhance TGF-{beta}-mediated suppression of trinitrobenzene sulfonic acid-induced colitis via a mechanism that involved suppression of IL-12 and Th1 responses, which in turn favored expansion of TGF-{beta}-producing Th3 cells (66).

The finding that naive T cells can in some circumstances be controlled by CD4+CD25+ TR cells via an IL-10-independent mechanism raises the question of why anti-IL-10R mAb treatment induces colitis in normal BALB/c mice. It would appear that the IL-10-independent control mechanism does not prevent priming of potentially colitogenic T cells in these animals, but is sufficient to prevent the development of colitis in SCID recipients following transfer of naive T cells. One possible explanation is that the IL-10-independent regulation is somewhat leaky such that in a normal mouse, some T cells become primed and are then able to induce pathology when IL-10 activity is removed. If this is so, then the IL-10-dependent mechanism would represent a second layer of regulation able to suppress primed T cell responses. However, when colitis is induced by transfer of naive cells, the IL-10-independent mechanism is sufficient to limit the proliferative expansion of potentially colitogenic cells to the extent that the mice fail to develop disease. Another and not mutually exclusive possibility is that in a normal mouse, only T cells with higher affinity for enteric Ag will be primed, such that those remaining in the naive T cell pool will be expressing lower affinity TCRs. Thus, transfer of the lower affinity cells that remain in the naive T cell pool is able to cause colitis in the absence of TR and yet remains controllable by TR even in the absence of IL-10. Clearly, additional experiments are required to identify the factors that control the requirement for IL-10 in TR cell-mediated control of pathologic T cell responses. The complexity is revealed by a recent report in which it was found that control of the immune response to Leishmania major by CD4+CD25+TR cells involved both IL-10-dependent and IL-10-independent mechanisms (67).

Therapeutic use of TR cells will require that they suppress Ag-experienced T cell responses and reverse established disease. The finding that control of Ag-experienced colitogenic T cells by TR cells is dependent on IL-10 suggests that production or induction of IL-10 may be a key factor in the therapeutic potential of these cells.


   Acknowledgments
 
We thank Drs. D. Mason, O. Annacker, K. Maloy, and L. Stephens for critical review of this manuscript. We also are indebted to N. Rust for excellent technical assistance with cell sorting, S. Biddolph for processing of histological samples, and C. Hetherington and staff for care of experimental animals. We gratefully acknowledge Dr. N. Glaichenhaus for the provision of GF mice, and Dr. V. Malmstrom for performing thymectomies.


   Footnotes
 
1 This work was funded by the Wellcome Trust (to C.A.). S.R. contributed equally to this study. Back

2 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}path.ox.ac.uk Back

3 Abbreviations used in this paper: IBD, inflammatory bowel disease; GF, germfree; LP, lamina propria; MLN, mesenteric lymph node; RAG, recombination-activating gene; RTE, recent thymic emigrant; SPF, specific pathogen free; TR, regulatory T; wt, wild type. Back

Received for publication February 13, 2003. Accepted for publication May 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Blumberg, R. S., L. J. Saubermann, W. Strober. 1999. Animal models of mucosal inflammation and their relation to human inflammatory bowel disease. Curr. Opin. Immunol. 11:648.[Medline]
  2. Powrie, F., M. W. Leach. 2001. Experimental models of inflammatory bowel disease. A. N. Theofilopoulos, and C. A. Bona, eds. The Molecular Pathology of Autoimmune Diseases Taylor & Francis, New York.
  3. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. Barcomb Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhighCD4+ T cells. Immunity 1:553.[Medline]
  4. Duchmann, R., E. Schmitt, P. Knolle, K. H. Meyer zum Buschenfelde, M. Neurath. 1996. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur. J. Immunol. 26:934.[Medline]
  5. Cong, Y., S. L. Brandwein, R. P. McCabe, A. Lazenby, E. H. Birkenmeier, J. P. Sundberg, C. O. Elson. 1998. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease. J. Exp. Med. 187:855.[Abstract/Free Full Text]
  6. Sellon, R. K., S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, E. Balish, D. M. Rennick, R. B. Sartor. 1998. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect. Immun. 66:5224.[Abstract/Free Full Text]
  7. Brimnes, J., J. Reimann, M. Nissen, M. Claesson. 2001. Enteric bacterial antigens activate CD4+ T cells from scid mice with inflammatory bowel disease. Eur. J. Immunol. 31:23.[Medline]
  8. Breese, E., C. P. Braegger, C. J. Corrigan, J. A. Walker-Smith, T. T. MacDonald. 1993. Interleukin-2- and interferon-{gamma}-secreting T cells in normal and diseased human intestinal mucosa. Immunology 78:127.[Medline]
  9. Fuss, I. J., M. Neurath, M. Boirivant, J. S. Klein, C. de la Motte, S. A. Strong, C. Fiocchi, W. Strober. 1996. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease: Crohn’s disease LP cells manifest increased secretion of IFN-{gamma}, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 157:1261.[Abstract]
  10. Duchmann, R., I. Kaiser, E. Hermann, W. Mayet, K. Ewe, K. H. Meyer zum Buschenfelde. 1995. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin. Exp. Immunol. 102:448.[Medline]
  11. Morrissey, P. J., K. Charrier, S. Braddy, D. Liggitt, J. D. Watson. 1993. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178:237.[Abstract/Free Full Text]
  12. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
  13. Simpson, S. J., S. Shah, M. Comiskey, Y. P. de Jong, B. Wang, E. Mizoguchi, A. K. Bhan, C. Terhorst. 1998. T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon {gamma} expression by T cells. J. Exp. Med. 187:1225.[Abstract/Free Full Text]
  14. Claesson, M. H., S. Bregenholt, K. Bonhagen, S. Thoma, P. Moller, M. J. Grusby, F. Leithauser, M. H. Nissen, J. Reimann. 1999. Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype. J. Immunol. 162:3702.[Abstract/Free Full Text]
  15. Malmstrom, V., D. Shipton, B. Singh, A. Al-Shamkhani, M. J. Puklavec, A. N. Barclay, F. Powrie. 2001. CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice. J. Immunol. 166:6972.[Abstract/Free Full Text]
  16. Aranda, R., B. C. Sydora, P. L. McAllister, S. W. Binder, H. Y. Yang, S. R. Targan, M. Kronenberg. 1997. Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients. J. Immunol. 158:3464.[Abstract]
  17. Powrie, F., S. Mauze, R. L. Coffman. 1997. CD4+ T cells in the regulation of inflammatory responses in the intestine. Res. Immunol. 148:576.[Medline]
  18. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  19. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract/Free Full Text]
  20. Stephens, L. A., D. Mason. 2000. CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25+ and CD25- subpopulations. J. Immunol. 165:3105.[Abstract/Free Full Text]
  21. Hara, M., C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell, P. J. Morris, F. Powrie, K. J. Wood. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789.[Abstract/Free Full Text]
  22. Shimizu, J., S. Yamazaki, S. Sakaguchi. 1999. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163:5211.[Abstract/Free Full Text]
  23. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract/Free Full Text]
  24. Thornton, A. E., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  25. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, A. Bandeira. 2001. CD25+CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.[Abstract/Free Full Text]
  26. Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immun. 2:816.[Medline]
  27. Shevach, E. M.. 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389.[Medline]
  28. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor-{beta} but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.[Abstract/Free Full Text]
  29. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190:995.[Abstract/Free Full Text]
  30. Suri-Payer, E., H. Cantor. 2001. Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4+CD25+ T cells. J. Autoimmun. 16:115.[Medline]
  31. Singh, B., S. Read, C. Asseman, V. Malmstrom, C. Mottet, L. Stephens, R. Stepankova, H. Tlaskalova, F. Powrie. 2001. Control intestinal inflammation by regulatory T cells. Immunol. Rev. 182:190.[Medline]
  32. Liu, Z., K. Geboes, P. Hellings, P. Maerten, H. Heremans, P. Vandenberghe, L. Boon, P. van Kooten, P. Rutgeerts, J. L. Ceuppens. 2001. B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis. J. Immunol. 167:1830.[Abstract/Free Full Text]
  33. Li, X., J. G. Fox, M. T. Whary, L. Yan, B. Shames, Z. Zhao. 1998. SCID/NCr mice naturally infected with Helicobacter hepaticus develop progressive hepatitis, proliferative typhlitis, and colitis. Infect. Immun. 66:5477.[Abstract/Free Full Text]
  34. Monaco, A. P., M. L. Wood, J. G. Gray, P. S. Russell. 1966. Studies on heterologous anti-lymphocyte serum in mice. II. Effect on the immune response. J. Immunol. 96:229.[Abstract/Free Full Text]
  35. Cobbold, S. P., A. Jayasuriya, A. Nash, T. D. Prospero, H. Waldmann. 1984. Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature 312:548.[Medline]
  36. Coffman, R. L.. 1982. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B cell development. Immunol. Rev. 69:5.[Medline]
  37. O’Farrell, A. M., Y. Liu, K. W. Moore, A. L. Mui. 1998. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways. EMBO J. 17:1006.[Medline]
  38. Openshaw, P., E. E. Murphy, N. A. Hosken, V. Maino, K. Davis, K. Murphy, A. O’Garra. 1995. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182:1357.[Abstract/Free Full Text]
  39. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263.[Medline]
  40. Davidson, N. J., M. M. Fort, W. Muller, M. W. Leach, D. M. Rennick. 2000. Chronic colitis in IL-10-/- mice: insufficient counter regulation of a Th1 response. Int. Rev. Immunol. 19:91.[Medline]
  41. Kullberg, M. C., J. M. Ward, P. L. Gorelick, P. Caspar, S. Hieny, A. Cheever, D. Jankovic, A. Sher. 1998. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and {gamma} interferon-dependent mechanism. Infect. Immun. 66:5157.[Abstract/Free Full Text]
  42. Bottomly, K., M. Luqman, L. Greenbaum, S. Carding, J. West, T. Pasqualini, D. B. Murphy. 1989. A monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different cytokines. Eur. J. Immunol. 19:617.[Medline]
  43. Bell, E. B.. 1992. Function of CD4 T cell subsets in vivo: expression of CD45R isoforms. Semin. Immunol. 4:43.[Medline]
  44. Scollay, R., A. Wilson, K. Shortman. 1984. Thymus cell migration: analysis of thymus emigrants with markers that distinguish medullary thymocytes from peripheral T cells. J. Immunol. 132:1089.[Abstract]
  45. Gabor, M. J., D. I. Godfrey, R. Scollay. 1997. Recent thymic emigrants are distinct from most medullary thymocytes. Eur. J. Immunol. 27:2010.[Medline]
  46. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18:423.[Medline]
  47. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda, T. Takahashi. 2001. Immunologic tolerance maintained by CD25CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182:18.[Medline]
  48. Birkeland, M. L., P. Johnson, I. S. Trowbridge, E. Pure. 1989. Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation. Proc. Natl. Acad. Sci. USA 86:6734.[Abstract/Free Full Text]
  49. Morrissey, P. J., K. Charrier. 1994. Induction of wasting disease in SCID mice by the transfer of normal CD4+CD45RBhigh T cells and the regulation of this autoreactivity by CD4+CD45RBlow T cells. Res. Immunol. 145:357.[Medline]
  50. Powrie, F., R. Correa-Oliveira, S. Mauze, R. L. Coffman. 1994. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract/Free Full Text]
  51. Furtado, G. C., D. Olivares-Villagomez, M. A. Curotto De Lafaille, A. K. Wensky, J. A. Latkowski, J. J. Lafaille. 2001. Regulatory T cells in spontaneous autoimmune encephalomyelitis. Immunol. Rev. 182:122.[Medline]
  52. Nakamura, K., A. Kitani, W. Strober. 2001. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor {beta}. J. Exp. Med. 194:629.[Abstract/Free Full Text]
  53. Kullberg, M. C., D. Jankovic, P. L. Gorelick, P. Caspar, J. J. Letterio, A. W. Cheever, A. Sher. 2002. Bacteria-triggered CD4+ T regulatory cells suppress Helicobacter hepaticus-induced colitis. J. Exp. Med. 196:505.[Abstract/Free Full Text]
  54. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J. Exp. Med. 184:19.[Abstract/Free Full Text]
  55. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, A. O’Garra. 2002. In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195:603.[Abstract/Free Full Text]
  56. Oida, T., X. Zhang, M. Goto, S. Hachimura, M. Totsuka, S. Kaminogawa, H. L. Weiner. 2003. CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-{beta}-dependent mechanism. J. Immunol. 170:2516.[Abstract/Free Full Text]
  57. Cottrez, F., H. Groux. 2001. Regulation of TGF-{beta} response during T cell activation is modulated by IL-10. J. Immunol. 167:773.[Abstract/Free Full Text]
  58. Almeida, A. R., N. Legrand, M. Papiernik, A. A. Freitas. 2002. Homeostasis of peripheral CD4+ T cells: IL-2R{alpha} and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J. Immunol. 169:4850.[Abstract/Free Full Text]
  59. Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, F. Powrie. 2003. CD4+CD25+ regulatory T cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197:111.[Abstract/Free Full Text]
  60. Gorham, J. D., M. L. Guler, D. Fenoglio, U. Gubler, K. M. Murphy. 1998. Low dose TGF-{beta} attenuates IL-12 responsiveness in murine Th cells. J. Immunol. 161:1664.[Abstract/Free Full Text]
  61. Ludviksson, B. R., D. Seegers, A. S. Resnick, W. Strober. 2000. The effect of TGF-{beta}1 on immune responses of naive versus memory CD4+ Th1/Th2 T cells. Eur. J. Immunol. 30:2101.[Medline]
  62. Gorelik, L., S. Constant, R. A. Flavell. 2002. Mechanism of transforming growth factor {beta}-induced inhibition of T helper type 1 differentiation. J. Exp. Med. 195:1499.[Abstract/Free Full Text]
  63. D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon- {gamma} production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041.[Abstract/Free Full Text]
  64. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, G. Schuler. 1996. High level IL-12 production by murine dendritic cells: up-regulation via MHC class II and CD40 molecules and down-regulation by IL-4 and IL-10. J. Exp. Med. 184:741.[Abstract/Free Full Text]
  65. Kitani, A., I. J. Fuss, K. Nakamura, O. M. Schwartz, T. Usui, W. Strober. 2000. Treatment of experimental (Trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-{beta}1 plasmid: TGF-{beta}1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor {beta}2 chain down-regulation. J. Exp. Med. 192:41.[Abstract/Free Full Text]
  66. Fuss, I. J., M. Boirivant, B. Lacy, W. Strober. 2002. The interrelated roles of TGF-{beta} and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900.[Abstract/Free Full Text]
  67. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502.[Medline]



This article has been cited by other articles:


Home page
 Sci SignalHome page
U. Schenk, A. M. Westendorf, E. Radaelli, A. Casati, M. Ferro, M. Fumagalli, C. Verderio, J. Buer, E. Scanziani, and F. Grassi
Purinergic Control of T Cell Activation by ATP Released Through Pannexin-1 Hemichannels
Sci. Signal., September 30, 2008; 1(39): ra6 - ra6.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Zhu and W. E. Paul
CD4 T cells: fates, functions, and faults
Blood, September 1, 2008; 112(5): 1557 - 1569.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. J. Winstead, J. M. Fraser, and A. Khoruts
Regulatory CD4+CD25+Foxp3+ T Cells Selectively Inhibit the Spontaneous Form of Lymphopenia-Induced Proliferation of Naive T Cells
J. Immunol., June 1, 2008; 180(11): 7305 - 7317.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
I. Dotan, M. Allez, A. Nakazawa, J. Brimnes, M. Schulder-Katz, and L. Mayer
Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-{gamma}
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1630 - G1640.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
H. Braat, P. McGuirk, F. J W Ten Kate, I. Huibregtse, P. J Dunne, D. W Hommes, S. J H Van Deventer, and K. H G Mills
Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule
Gut, March 1, 2007; 56(3): 351 - 357.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. H. Uhlig, J. Coombes, C. Mottet, A. Izcue, C. Thompson, A. Fanger, A. Tannapfel, J. D. Fontenot, F. Ramsdell, and F. Powrie
Characterization of Foxp3+CD4+CD25+ and IL-10-Secreting CD4+CD25+ T Cells during Cure of Colitis
J. Immunol., November 1, 2006; 177(9): 5852 - 5860.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Read, R. Greenwald, A. Izcue, N. Robinson, D. Mandelbrot, L. Francisco, A. H. Sharpe, and F. Powrie
Blockade of CTLA-4 on CD4+CD25+ Regulatory T Cells Abrogates Their Function In Vivo
J. Immunol., October 1, 2006; 177(7): 4376 - 4383.
[Abstract] [Full Text] [PDF]