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* Division of Gastroenterology and Hepatology and
Department of Pathology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1)
cells. Bir Tr1 cells proliferated poorly, but their proliferation was
dependent on CD28-B7 interactions and was MHC class II-restricted.
Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and
cotransfer of Bir Tr1 T cells with pathogenic Bir CD4+ Th1
cells prevented colitis. Bir Tr1 cells inhibited proliferation and
IFN- production of a CBA-specific Th1 cell line in vitro.
Such inhibition was partly due to IL-10 and TGF
1, but cognate
interactions with either APCs or Th1 cells were also involved. Normal
intestinal lamina propria CD4+ T cells had Tr1-like
activity when stimulated with CBA-pulsed APCs. We conclude that
CD4+ T cells with the properties of Tr1 cells are present
in the intestinal lamina propria and hypothesize that these cells
maintain intestinal immune homeostasis to the enteric
flora. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strong support for the concept that mucosal immune responses to
bacterial flora are tightly regulated come from a number of
experimental systems showing that altered regulation of intestinal T
cell function can result in chronic intestinal inflammation. This has
been evident in a number of newly developed induced mutant and other
mouse models, including IL-2 deficiency (12), IL-10
deficiency (13), and a model in which normal
CD4+, CD45RBhigh T cells
are transferred into syngeneic SCID or RAG-/-
recipients (14, 15) in whom they cause colitis, wasting,
and death. In the latter model, cotransfer of the reciprocal
CD4+, CD45RBlow T cell
subset prevents disease, due to the presence in this subset of a
regulatory T cell population that produces IL-10 and TGF (16, 17). These results indicate that both potentially pathogenic and
regulatory T cells are present in normal mice. The stimulus driving
such pathogenic and regulatory T cells comes from the enteric bacterial
flora because no disease occurs when the animals are raised under
germfree conditions (18, 19) and the disease is
substantially ameliorated when the animals are raised under specific
pathogen-free conditions (20, 21).
The bacterial flora is a very complex assortment of Ags, mitogens, and immunomodulators, and in most models it remains unknown which of these factors might be stimulating the pathogenic T cell response. However, in previous work on a spontaneous model of colitis, the C3H/HeJBir mouse, pathogenic T cells responsive to protein Ags of the bacterial flora caused colitis after transfer to histocompatible C3H/HeSnJ scid/scid recipients. Transfer of normal C3H/HeJ CD4+ T cells did not cause disease. Multiple bacterial-reactive CD4+ T cell lines have been derived from C3H/HeJBir mice (22). Most of these are Th1 cells and cause colitis when transferred into C3H-SCID recipients. Interestingly, the adult C3H/HeJBir donors of these pathogenic T cells were not ill, suggesting that regulatory cells were controlling pathogenic T cell activity and preventing disease in vivo. In this study, we report on the presence of CD4+ regulatory T cells in C3H/HeJBir mice, T cells with the characteristics of the T regulatory-1 (Tr1) subset, recently described in a DO11.10 TCR transgenic system (23). Bacterial-reactive Tr1 cells were able to inhibit pathogenic memory Th1 cells in vitro and in vivo using multiple mechanisms. Moreover, CD4+ T cells with the properties of Tr1 cells were identified in the normal mouse intestinal LP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeJBir, C3H/HeJ, and C3H/HeSnJ-Pkrdcscid/Pkrdcscid (C3H-SCID) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the University of Alabama (Birmingham, AL) animal facility. All studies were approved by the Animal Care and Use Committee of the University of Alabama.
Reagents and Abs
RPMI 1640 and FBS were purchased from Life Technologies (Long
Island, NY). 2-ME, HEPES, L-glutamine, and sodium pyruvate
were purchased from Sigma-Aldrich (St. Louis, MO). mAbs to CD4, CD8,
ICAM-1, 7 integrin, CD44, L-selectin, B7.1
(1G10), B7.2 (GL1), I-Ab (25-9-17),
I-Ak (11-5.2), and isotype control Abs were
purchased from BD PharMingen (San Diego, CA). Anti-TGF1 (1D11) was
purchased from R&D Systems (Minneapolis, MN). Anti-IL-10R1 (1B1.2) was
a gift from Dr. K. Moore (DNAX Research Institute, Palo Alto, CA) and
anti-CD40 ligand (CD40L) (MR1) was kindly provided by Dr. B.
Kelsoe (National Institutes of Health, Bethesda, MD).
Preparation of enteric bacterial Ags
Cecal bacterial Ags (CBA) were prepared as previously described (24). Briefly, C3H/HeJ or C3H-SCID mice were sacrificed and their ceca removed. The ceca were opened and placed in 1 ml of PBS. Cecal 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 rpm in a MiniBead Beater (Biospec Products, Bartlesville, OK) for 3 min and then placed on ice. The glass beads and unlysed cells were removed by centrifugation at 5000 x g for 5 min. The lysates were filter-sterilized through a 0.2-µ syringe filter.
Establishment and maintenance of enteric bacterial Ag-specific Tr1, Th1, and Th2 cells
C3H/HeJBir spleen and mesenteric lymph nodes (MLN) were removed from groups of four to five mice and placed into cell suspension by straining through a small mesh sieve as described previously (25). After two washes, the cells were passed through a nylon wool column to obtain T cells as previously described (26). The column-passed cells were washed twice and treated with rat IgM anti-CD8 mAb (TIB 211; American Type Culture Collection, Manassas, VA) supernatant (1 ml supernatant/1 x 107 cells) for 30 min on ice. After washing three times, magnetic beads coated with anti-rat IgM were added to the cells (BioMag, Cambridge, MA) and incubated for 30 min on ice. After passing through a magnet, CD4+ T cells were collected and reconstituted at 4 x 106 cells per ml in complete RPMI medium containing 10% FCS for use in cell culture.
For APCs, spleen cells from C3H/HeJ mice were isolated and treated with 200 µg/ml enteric bacterial Ag at 2 x 107 cells/5 ml in a 15-ml tube overnight at 37°C. After washing twice, the cells were reconstituted at 4 x 106 cells per ml in complete medium containing RPMI 1640 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml penicillin, and 10 µg/ml streptomycin for use in cell culture. These APCs (CBA-APC) were irradiated with 3000 R before being added to T cell cultures. In some experiments, APCs were pulsed with 200 µg/ml proteins extracted from food pellets, or from an intestinal epithelial cell line, Mode K (a gift from Dr. D. Kaiserlian, Batiment Institut Pasteur, Lyon, France).
To generate Tr1 cells, 4 x 106 T cells and 4 x 106 CBA-pulsed APCs were cultured per well in 24-well plates in the presence or absence of 10 ng/ml IL-10. T cells were restimulated with freshly prepared CBA-APCs in the presence of IL-10 at intervals of 10–14 days.
Th1 and Th2 cells were generated in vitro from CD4+ cells. Briefly, purified CD4+ T cells from MLN of C3H/NeJBir mice were cultured at a ratio of 1:1 with irradiated CBA-pulsed APC. The addition of 50 U/ml IL-12 (R&D Systems) and 10 µg/ml anti-IL-4 (11B11) was used to generated Th1 cells, while 1000 U/ml IL-4 (R&D Systems) and 10 µg/ml anti-IL-12 (C17.8) were used to generated Th2 cells. Recovered cells were washed and restimulated with CBA-APC at the same conditions described above every 2 wk.
Preparation of LP lymphocytes
LP lymphocytes were isolated as previously described
(27). Briefly, the intestines from 10 mice were removed,
the Peyer’s patches were carefully excised, and the remaining
intestine was kept on ice until used. For removal of epithelial cells
and intraepithelial lymphocytes, the intestines were washed, cut into
small pieces, and then the pieces were incubated with calcium- and
magnesium-free HBSS supplemented with 2% bovine calf serum and 5 mM
EDTA (Sigma-Aldrich) on a magnetic stirrer at 37°C for 30 min. This
process was repeated three times. The tissues were then incubated with
RPMI 1640 containing 2% bovine calf serum, antibiotics, 25 mM HEPES
and 1.5 mg/ml Dispase (Roche Diagnostics, Indianapolis, IN) for
30 min at 37°C with stirring. The liberated cells were collected by
passage through a stainless steel sieve. The digestion was repeated
three times. The isolated cells were pooled together and separated on a
40/75% discontinuous Percoll gradient (Pharmacia, Piscataway, NJ)
centrifuged at 600 x g, 25°C for 20 min. The cell
yield was typically 
2 x 106 lymphocytes
per mouse with >90% cell viability. After two washes, the cells were
passed through a nylon wool column to obtain T cells as previously
described (26). The column-passed cells were washed twice
and depleted of CD8+ T cells as described in the
preceding section.
Assay of Ag-specific proliferation of T cells
T cell lines generated as above were cultured at 2 x 105 cells/well in triplicate in the presence of 2 x 105 Ag-pulsed APC/well in 96-well flat-bottom tissue culture plates (Falcon 3072; BD Labware, Franklin Lakes, NJ) at 37°C in 5% CO2 humidified air. After 3 days of culture, 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to each culture for the last 16–20 h of the incubation period. The cells were harvested on glass fiber filters on a PHD cell harvester (Cambridge Technology, Watertown, MA). Proliferation was measured as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta-scintillation counting (Beckman Instruments, Palo Alto, CA) of the harvested samples. Data were expressed as mean cpm ± SD.
Cytokine assays
T cell lines were cultured in the presence of APC pretreated
with various Ags as described above. Culture supernatants were
collected at different times and pooled together for assay.
Supernatants collected after 24 h of culture were used for IL-2
assay and the supernatants collected at 48–72 h of culture were used
for IL-4, IL-10, and IFN- assays. To measure IL-12 production,
C3H-SCID spleen APC were cultured with T cell lines under different
conditions as described in Table III
text, and the supernatant
was collected at 48 h of culture.
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, R4-6A2/XMG1.2; IL-12, C17.8/C15.6. Intracellular analysis of cytokine production by flow cytometry
Intracellular cytokines were detected by flow cytometry as
previously described (29). Briefly, LP
CD4+ T cells were stimulated with CBA-pulsed APC
in the presence of anti-IL-10R mAb or control Ab. Seven days later,
LP CD4+ T cells were restimulated with
anti-CD3 (10 µg/ml) and anti-CD28 (10 µg/ml) in the
presence of anti-IL-10R mAb or control Ab. After 2 h of
culture, brefeldin A (10 µg/ml; BD PharMingen) was added. After a
total 5-h culture, the cells were collected, washed in PBS, and stained
with anti-CD4 mAb. After fixation with 2% formaldehyde, the cells
were permeabilized by incubation in PBS supplemented with 2% FCS and
0.5% saponin (BD PharMingen). Permeabilized cells were stained with
PE-labeled anti-IL-4, or anti-IFN- and APC-labeled
anti-IL-10 mAbs (BD PharMingen). After washing, the cells were
analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View,
CA), and data were analyzed with CellQuest software (BD Biosciences). A
PE- or APC-labeled control mAb of the same isotype was used as a
negative control in all experiments.
Adoptive transfer and evaluation of colitis
For CD4+ Bir T cell transfers, T cell lines were harvested 5 days after restimulation with CBA-APCs. Dead cells were removed by centrifugation over lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada), then 2 x 106 T cells were transferred i.v. into C3H-SCID recipients. Three months later, the recipients were killed and the cecum and the proximal, middle, and distal portions of colon were fixed in formalin. If animals looked ill and had lost >20% of initial body weight they were sacrificed at that point along with a randomly selected animal from the control group. Fixed tissues were embedded in paraffin, and sections were stained with H&E for histologic examination. All slides were read by an experienced pathologist (A.L.) without knowledge of their origin.
Statistics
The results were expressed as the mean ± SD. The significance of the difference between means was determined by the Mann-Whitney test, and differences were considered statistically significant with p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate an enteric bacterial Ag-specific Tr1 cell line, MLN
CD4+ T cells of C3H/HeJBir mice were repeatedly
stimulated with CBA-APC in the presence of IL-10. Such cultures expand
memory T cell subsets that have been primed in vivo. After several
cycles of stimulation, cytokine production in the culture supernatants
was measured. As shown in Fig. 1A, the resulting T cells
(Tr1) produced high levels of IL-10, and low amounts of IL-4 and
IFN-, with no IL-2 production, and small amounts of TGF,
consistent with the cytokine profile of Tr1 cells described by Groux et
al. (23). These cells proliferated poorly to CBA-APC
stimulation compared with the Th1 or Th2 cells with the same CBA
specificity (Fig. 1B). Intracellular cytokine staining
showed that most Tr1 cells produced only IL-10 (Fig. 1C). To
determine the Ag specificity of CBA-specific Tr1 cells, their ability
to proliferate was measured after stimulation with APCs pulsed with
CBA, food, epithelial, or unrelated Ags. They responded to CBA-pulsed
APCs, but not to APCs pulsed with epithelial cell, food, or unrelated
protein Ags such as OVA (Table I).
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Growth factors and costimulatory requirement of Tr1 cells
As shown in Fig. 1
B, Tr1 cells proliferated poorly to
CBA stimulation. Various Abs against different cytokines were added
into the Bir Tr1 cell culture separately or in different combinations.
Addition of anti-IL-2 did not affect Tr1 cell growth. Anti-IL-10
affected cell growth slightly or not at all. In contrast, anti-IL-4
Ab inhibited Tr1 cell growth, but never completely (Fig. 2A), indicating that IL-4 is
partially responsible for Tr1 cell growth. Addition of exogenous
recombinant IL-2, IL-4, or IL-10 had no effect on proliferation of Tr1
cells (data not shown).
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B).
Tr1 inhibition of pathogenic T cell proliferation and IFN-
production in vitro
We have established previously pathogenic CBA-reactive Th1 cell
lines from C3H/HeJBir mice that cause colitis in all C3H-SCID
recipients upon transfer (22). Tr1 cells were irradiated
and added to the cultures of pathogenic Th1 cells plus CBA-APC. Then T
cell proliferation was measured after a 5-day culture. As shown in
Table II, Th1 cells responded strongly to
CBA stimulation, and this Th1 cell response was inhibited by the
presence of Tr1. Similar results were obtained when IFN- production
was measured; Th1 cells produced high amounts of IFN-, and this
IFN- production was inhibited by Tr1 cells (data not shown). To
determine whether cytokines produced by Tr1 cells caused such
inhibition, Abs against different cytokines were added into these
Th1-Bir 8 cultures. Addition of anti-IL-4 had no effects on
inhibition by Tr1 cells. Addition of anti-IL-10R1, anti-TGF1
or both Abs reversed Tr1 inhibition partially. Anti-IL-10R1 had a
greater effect than did anti-TGF1.
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Ab completely blocked
Tr1 cell inhibition of pathogenic Th1 cell responses, it seemed
possible that Tr1 inhibition required cell contact as well as soluble
factors. To determine whether cell contact was required, a transwell
culture system was used. Tr1 cells were added into the upper wells with
CBA-APC and pathogenic Th1 cells were added into the lower wells with
CBA-APC. Such cultures were compared with those in which both Tr1 and
Th1 cells were cultured together with CBA-APCs in the lower wells. Tr1
cells in the upper wells were able to inhibit Th1 cell response to CBA
stimulation in the lower wells, but to a lesser extent than when Tr1
cells were added together with Th1 in the same lower wells (Fig. 3A), indicating that Tr1 cell
inhibition of pathogenic Th1 cell responses required some cell contact.
In additional experiments, we activated Tr1 cells with CBA-pulsed APCs
for 2 days in vitro, then fixed them in paraformaldehyde. Such
paraformaldehyde-fixed Tr1 cells inhibited the Th1 cell response to
CBA-APC, but less so than did unfixed Tr1 cells (Fig. 3B).
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Pathogenic Th1 cells reactive to enteric bacterial Ags induce
IL-12 production by APC, which is essential to the induction of colitis
by these pathogenic T cells (22). To determine whether Tr1
cells inhibited APC IL-12 production, they were added to cultures of
Th1 cells with CBA-APC. Two days later, supernatants were collected and
IL-12 p40 production was measured by ELISA. Th1 cells strongly induced
IL-12 production by CBA-APC. Such IL-12 production was inhibited by Tr1
cells (Table III). Interestingly,
addition of anti-IL-10R1 Ab reversed the Tr1 cell inhibition of APC
IL-12 production, indicating that the Tr1 inhibition of APC IL-12
production was mediated by IL-10. Anti-TGF1 did not reverse the
inhibition of IL-12 by Tr1 cells (data not shown).
Tr1 cells prevent colitis induced by pathogenic Th1 cells
To investigate the ability of CBA-specific Tr1 cells to prevent
colitis induced by pathogenic Th1 cells in vivo, groups of four
C3H-SCID mice were given either 1 x 106 Tr1
cells, 1 x 106 pathogenic Th1 cells, or
both together. Three months after cell transfer, the recipients were
sacrificed and the histopathology of the small intestine, cecum, and
the proximal, middle, and distal portions of colon were examined. Bir
Tr1 cells did not cause colitis in any recipients. All four recipients
of pathogenic Bir Th1 cells developed colitis (Fig. 4B). The lesions in the
recipients were focal and prominent in the cecum and proximal colon, as
previously described (22). The small intestine of these
recipients did not show any inflammation. Interestingly, none of
recipients that received both Tr1 cells and pathogenic Th1 cells
developed disease (Fig. 4A) indicating that this Tr1 subset
could inhibit the potent pathogenic effects of an activated, memory Th1
cell line in vivo.
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LP CD4+ T cells from normal C3H/HeJ mice
were isolated and added into cultures of pathogenic Th1 cells in the
presence of CBA-pulsed APC or unpulsed APCs plus anti-CD3/CD28. T
cell proliferation and IFN- production were measured. As shown in
Table IV, T cell proliferation was
inhibited by LP CD4+ T cells in response to
CBA-APC. In contrast, LP CD4+ T cells did not
inhibit the cell response to anti-CD3/CD28 stimulation, suggesting
that LP CD4+ T cells inhibit Th1 cells indirectly
via effects on APCs. Interestingly, addition of anti-IL-10R1 mAb,
but not anti-IL-4 mAb, partially abrogated LP
CD4+ T cell inhibition of the Th1 response,
consistent with IL-10 being involved in LP CD4+ T
cell inhibition.
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LP CD4+ T cells from C3H/HeJ mice were
isolated and stimulated with CBA-pulsed APCs. Culture supernatants were
collected at day 1 for measuring IL-2 and at day 3 for measuring IL-4,
IL-10, and IFN-. As shown in Table V, only IL-10, but not IL-2, IL-4,
and IFN-, production was detected in culture supernatants.
Interestingly, when anti-IL10R1 mAb was added into culture, both
IL-10 and IFN- production were detected.
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, 10% of LP
CD4+ T cells were IL-10+,
and almost none of the LP CD4+ T cells were
IFN-+ (0.2%) or IL-4+
(0.3%). (Fig. 5, A and C) when stimulated with
enteric bacterial Ags. When anti-IL-10R1 mAb was added into
culture, 9.4% LP CD4+ T cells remained
IL-10+ but 4.8% were
IFN-+ (Fig. 5D) and still very few
were IL-4+ (0.5%) (Fig. 5B). Notably,
no LP CD4+ T cells coexpressed IL-10 and IFN-
(Fig. 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and no IL-2. More
importantly, when cotransferred with pathogenic Th1 cells reactive to
enteric bacterial Ags, these Tr1 cells prevented colitis, indicating
that Tr1 cells can function in vivo as regulatory cells.
The protective effect of regulatory T cells has been observed in
several experimental model systems. A variety of organ-specific
autoimmune diseases that arise in mice thymectomized at day 3 of life
can be prevented by administration of
CD4+CD25+ T cells
(31, 32). In thymectomized and irradiated adult
PVG.RT1u rats, the onset of autoimmune diabetes
can be prevented by transfer of
CD4+CD45RClow,
RT66 T cells (33, 34). In nonobese
diabetic female mice, thymectomy at weaning prevents the
generation of protective cells and thus accelerates the onset of
diabetes (35). CD4+ T cells from
nondiabetic young mice prevent the transfer of diabetes by splenocytes
from diabetic mice (36, 37).
CD4+
T cells also confer protection from
spontaneous experimental autoimmune encephalomyelitis
(38).
Thus, the existence of regulatory T cells capable of protecting against
autoimmune diseases, or allowing transplantation, has been supported by
data from several laboratories and it is likely that several types of
regulatory T cell subsets exist. Recently, T cell subsets have been
identified that have regulatory function in the mucosae. One of these,
denoted Tr1, produces high amounts of IL-10; another, denoted Th3,
produces TGF1. Both subsets appear to be involved in mucosal immune
regulation in that deletion of genes encoding their effector molecules,
their receptors, or their critical signal transduction molecules all
result in chronic intestinal inflammation (13, 39, 40, 41, 42).
To study the mechanisms mediating Bir Tr1 cell inhibition of Th1 cells,
Tr1 cells were cocultured with pathogenic Th1 cells in vitro. Bir Tr1
cells substantially inhibited Th1 cell responses to enteric bacterial
Ags, in that both Th1 cell proliferation, as well as IFN- production
was decreased. This Bir Tr1 cell inhibition was mediated in part by
production of IL-10 and TGF, in that addition of anti-IL-10R1
mAb or anti-TGF1 mAb partially blocked the Bir Tr1 inhibition.
Interestingly, addition of both anti-IL-10R1 and anti-TGF1
did not reverse the inhibition by Tr1 cells more than the addition of
either alone, indicating that IL-10 and TGF1 did not work
synergistically. This is similar to the CD45RBlow
regulatory T cell inhibition of colitis induced by
CD45RBhighCD4+ T cells in
that this inhibition was abrogated by either anti-IL-10R1 mAb or
anti-TGF1 mAb (16, 17).
Yet a third molecule involved in the Bir Tr1 cell regulation of Th1
responses appears to be a cell surface molecule. The lack of complete
reversal of inhibition by anti-IL-10R1 and anti-TGF1 alone
or in combination indicated that these two inhibitory cytokines could
not account for the entire inhibitory effect. A transwell experiment
confirmed the importance of cell contact in that inhibition was less
when the regulatory and effector cells were not in contact in the same
wells. More convincingly, Bir Tr1 cells that had been activated with
CBA-APC and then fixed were still able to inhibit Th1 cells in vitro
(Fig. 3B). The cell surface activity involved in Bir Tr1
cell function is not yet identified. The involvement of cytokines
(IL-10, TGF) as well as cognate interactions by Bir Tr1 cells in
vitro is different from the effects of
CD25+CD4+ T regulatory
cells (31, 32, 43, 44, 45), which require cell-cell contact,
but not soluble factors when measured in vitro, even though in vivo the
inhibition did involve TGF1 (45). There is evidence
that cell contact-dependent immunosuppression by
CD4+CD25+ regulatory T
cells is mediated by cell surface-bound TGF1 (45).
CTLA-4 had been shown to play an essential role in the function of
CD25+CD4+ regulatory cells
(43, 44). The role of CTLA-4 in Tr1 cell function remains
to be defined.
Bir Tr1 cells were specific for enteric bacterial Ags in that they proliferated to such enteric bacterial Ags, but not to food or epithelial Ags. The Bir Tr1 cell responses to enteric bacterial Ags were restricted by MHC class II, compatible with stimulation through the TCR. Tr1 proliferation was weak compared with Th1 and Th2 cell lines with similar reactivity. Interestingly, addition of either anti-B7.1 or anti-B7.2 Ab partially inhibited Bir Tr1 cell proliferation, but both together greatly inhibited it. In contrast, addition of anti-CD40L had no effect on Bir Tr1 cell proliferation, indicating that Tr1 cell proliferation required CD28-B7, but not CD40L-CD40, costimulation. Both pathogenic Bir Th1 lines (22) and Bir Tr1 cells (data not shown) were pauciclonal rather than polyclonal, and used TCRBV 6, 8, and 14 predominantly, the same TCRBV expressed by LP CD4 T cells in C3H/HeJBir mice (22). Whether these effector and regulatory T cells respond to the same or different bacterial Ags remains unknown.
Intestinal LP T cells have increased activation markers
(46), increased levels of cytokine gene expression
(47), and have a higher proportion of cells bearing the
CD45RO marker (48), consistent with their being highly
enriched for memory cells. Remarkably, little is known about T cells in
the mucosa that are reactive to Ags of the very abundant enteric
bacteria. We present data in this study that LP
CD4+ T cells contain regulatory T cells that
recognize enteric bacterial Ags. When added into enteric bacterial
Ag-specific Th1 cell cultures in the presence of CBA-APC, LP
CD4+ T cells inhibited Th1 cell proliferation as
well as IFN- production, partly due to secretion of IL-10. These
results are strikingly similar to the results obtained with Bir Tr1
cells and show that enteric bacterial Ag-specific
CD4+ cells with regulatory activity were present
in normal intestinal mucosae. Interestingly, upon enteric bacterial Ag
stimulation, both IL-10 and IFN- were detectable after IL-10
functional activity was blocked by addition of IL-10R-1 mAb.
Intracellular staining confirmed the above observation and clearly
showed that IL-10 and IFN- were produced by separate LP
CD4+ T cell subsets. These results are compatible
with previous work that identified both IL-10-producing and
IFN--producing CD4+ T cell subsets in the
intestinal LP of DO11.10 mice (49, 50). These data suggest
that both Tr1 and Th1 cells are present in the LP and that regulatory T
cells appear to play a dominant role in controlling mucosal immune
responses to enteric bacteria and preventing inflammation in the
mucosa.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Charles O. Elson, Division of Gastroenterology and Hepatology, University of Alabama, 633 Ziegler Research Building, 703 South 19th Street, Birmingham, AL 35294-0007. E-mail address: charles_elson{at}gasmac.dom.uab.edu
3 Abbreviations used in this paper: LP, lamina propria; Tr1, T regulatory-1; CD40L, CD40 ligand; CBA, cecal bacterial Ag; MLN, mesenteric lymph node.
Received for publication March 22, 2002. Accepted for publication September 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.-deficient mice fail to develop colitis in the absence of a microbial environment. Am. J. Pathol. 150:91.[Abstract]
1 gene results in multifocal inflammatory disease. Nature 359:693.[Medline]
1 null mutation in mice causes excessive inflammatory response and early death. Proc. Nat. Acad. Sci. USA 90:770.. EMBO J. 18:1280.[Medline]
. J. Exp. Med. 194:629. T cell receptor-transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene-specific antigen. Eur. J. Immunol. 27:1774.[Medline]
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 M Busse, T Traeger, C Potschke, A Billing, A Dummer, E Friebe, C Kiank, U Grunwald, R S Jack, C Schutt, et al. Detrimental role for CD4+ T lymphocytes in murine diffuse peritonitis due to inhibition of local bacterial elimination Gut, February 1, 2008; 57(2): 188 - 195. [Abstract] [Full Text] [PDF]
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 D. Noguchi, D. Wakita, M. Tajima, S. Ashino, Y. Iwakura, Y. Zhang, K. Chamoto, H. Kitamura, and T. Nishimura Blocking of IL-6 signaling pathway prevents CD4+ T cell-mediated colitis in a Th17-independent manner Int. Immunol., December 1, 2007; 19(12): 1431 - 1440. [Abstract] [Full Text] [PDF]
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J. Mestecky, M. W. Russell, and C. O. Elson Perspectives on Mucosal Vaccines: Is Mucosal Tolerance a Barrier? J. Immunol., November 1, 2007; 179(9): 5633 - 5638. [Abstract] [Full Text] [PDF]
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 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]
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G. Bamias, A. Okazawa, J. Rivera-Nieves, K. O. Arseneau, S. A. De La Rue, T. T. Pizarro, and F. Cominelli Commensal Bacteria Exacerbate Intestinal Inflammation but Are Not Essential for the Development of Murine Ileitis J. Immunol., February 1, 2007; 178(3): 1809 - 1818. [Abstract] [Full Text] [PDF]
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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]
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C. Gianfrani, M. K. Levings, C. Sartirana, G. Mazzarella, G. Barba, D. Zanzi, A. Camarca, G. Iaquinto, N. Giardullo, S. Auricchio, et al. Gliadin-Specific Type 1 Regulatory T Cells from the Intestinal Mucosa of Treated Celiac Patients Inhibit Pathogenic T Cells J. Immunol., September 15, 2006; 177(6): 4178 - 4186. [Abstract] [Full Text] [PDF]
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 W. Barchet, J. D. Price, M. Cella, M. Colonna, S. K. MacMillan, J. P. Cobb, P. A. Thompson, K. M. Murphy, J. P. Atkinson, and C. Kemper Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation Blood, February 15, 2006; 107(4): 1497 - 1504. [Abstract] [Full Text] [PDF]
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J. Brimnes, M. Allez, I. Dotan, L. Shao, A. Nakazawa, and L. Mayer Defects in CD8+ Regulatory T Cells in the Lamina Propria of Patients with Inflammatory Bowel Disease J. Immunol., May 1, 2005; 174(9): 5814 - 5822. [Abstract] [Full Text] [PDF]
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D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]
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 T. T. MacDonald and G. Monteleone Immunity, Inflammation, and Allergy in the Gut Science, March 25, 2005; 307(5717): 1920 - 1925. [Abstract] [Full Text] [PDF]
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Y. Cong, A. Konrad, N. Iqbal, R. D. Hatton, C. T. Weaver, and C. O. Elson Generation of Antigen-Specific, Foxp3-Expressing CD4+ Regulatory T Cells by Inhibition of APC Proteosome Function J. Immunol., March 1, 2005; 174(5): 2787 - 2795. [Abstract] [Full Text] [PDF]
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O. R. Millington, A. McI. Mowat, and P. Garside Induction of Bystander Suppression by Feeding Antigen Occurs despite Normal Clonal Expansion of the Bystander T Cell Population J. Immunol., November 15, 2004; 173(10): 6059 - 6064. [Abstract] [Full Text] [PDF]
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M. Akdis, J. Verhagen, A. Taylor, F. Karamloo, C. Karagiannidis, R. Crameri, S. Thunberg, G. Deniz, R. Valenta, H. Fiebig, et al. Immune Responses in Healthy and Allergic Individuals Are Characterized by a Fine Balance between Allergen-specific T Regulatory 1 and T Helper 2 Cells J. Exp. Med., June 7, 2004; 199(11): 1567 - 1575. [Abstract] [Full Text] [PDF]
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 M. Schultz, U. G. Strauch, H.-J. Linde, S. Watzl, F. Obermeier, C. Gottl, N. Dunger, N. Grunwald, J. Scholmerich, and H. C. Rath Preventive Effects of Escherichia coli Strain Nissle 1917 on Acute and Chronic Intestinal Inflammation in Two Different Murine Models of Colitis Clin. Vaccine Immunol., March 1, 2004; 11(2): 372 - 378. [Abstract] [Full Text] [PDF]
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A. Foussat, F. Cottrez, V. Brun, N. Fournier, J.-P. Breittmayer, and H. Groux A Comparative Study between T Regulatory Type 1 and CD4+CD25+ T Cells in the Control of Inflammation J. Immunol., November 15, 2003; 171(10): 5018 - 5026. [Abstract] [Full Text] [PDF]
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