Regulatory T cells modulate staphylococcal enterotoxin B-induced effector T-cell activation and acceleration of colitis.
ABSTRACT Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4(+) CD45RB(high) or CD4(+) CD45RB(high) plus CD4(+) CD45RB(low) T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4(+) CD45RB(high) and CD4(+) CD45RB(low) T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4(+) CD45RB(high) T cells alone. The latter was associated with an increase in the number of CD4(+) Vbeta8(+) T cells expressing CD69 and a significantly lower number of CD4(+) CD25(+) Foxp3(+) T cells. These changes were not observed in SCID mice reconstituted with both CD45RB(high) and CD45RB(low) T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4(+) CD45RB(high) T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.
- Gastroenterology 08/2000; 119(1):254-7. · 12.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Inflammatory bowel diseases (IBDs) are chronic debilitating conditions, which impair the patient's quality of life significantly. Among them, Crohn's disease and ulcerative colitis are idiopathic disorders for which an infective etiology has long been sought. Here, we present an opinion in support of the hypothesis that bacterial superantigens can participate in the initiation, exaggeration or reactivation of enteric inflammatory disease, at least in some patients. Although the identification of a specific pathogen responsible for IBD remains a worthy pursuit, an awareness of the response to bacterial products per se will be of value in providing a comprehensive understanding of enteric pathophysiological mechanisms and their potential role in IBDs.Trends in Immunology 10/2001; 22(9):497-501. · 9.49 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Although mechanisms operative in the induction and maintenance of specific, adaptive immunity, including 'cognate' B/T interactions, have been extensively studied and defined, we still know little about the mechanisms operative in developing and maintaining B- and T-cell dependent 'natural' immunity. Particularly, we are still rather ignorant concerning gut microbial/gut or systemic APC, T cell and B cell interactions that lead to lymphoid cell mediated 'natural' immunity: specific or broadly reactive, activation via TCR and BCR and/or via other receptors such as the TLR series, and whether T/B interactions are operative at this level? Here we will address: (1) the general role of gut microbes in the development and maintenance of the intestinal, humoral immune system; (2) the general role of gut microbes in the development of B1 cell mediated, 'natural' gut IgA and the dependence of these B1 cells on bystander T cell help; (3) the relative contributions of B1 versus B2 cells to gut 'natural' and specific IgA responses; (4) the role for particular 'normal' gut microbes in the initiation of inflammatory bowel diseases (IBD) in mice with a dysregulated immune system; and (5) the possible roles of gut microbes in facilitating oral tolerance, a mechanism likely operative in forestalling or ameliorating IBD. A central theme of this paper is to attempt to define the specificities of activated, functional CD4+ T cells in the gut for Ags of particular, usually benign gut microbes. We will also consider the still-unresolved issue of whether the contributions of B1-derived IgA in the gut to the 'natural' Ab pool are Ag-selected and driven to proliferation/differentiation or whether the main stimuli are not via BCRs but rather other receptors (TLRs, etc.). The main experimental approach has been to use antigen-free, germ-free, or gnotobiotic (mono- or oligo-associated with precisely known bacterial species) mice.Vaccine 03/2004; 22(7):805-11. · 3.49 Impact Factor
INFECTION AND IMMUNITY, Feb. 2009, p. 707–713
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 2
Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced
Effector T-Cell Activation and Acceleration of Colitis?
Armando Heriazon,1Pengfei Zhou,4Rajka Borojevic,3Katharina Foerster,1Catherine J. Streutker,2
Terry Ng,5and Kenneth Croitoru1*
Department of Medicine, Division of Gastroenterology, Mount Sinai Hospital, and Medical Sciences Division, University of Toronto,1
Department of Laboratory Medicine and Pathology, St. Michael’s Hospital and University of Toronto,2Toronto, Ontario, Canada;
Intestinal Disease Research Program, Department of Medicine, McMaster University, Hamilton, Ontario, Canada3;
Discovery Research Schering-Plough Biopharma, Palo Alto, California4; and University College Cork,
School of Medicine, Cork, Ireland5
Received 2 July 2008/Returned for modification 19 September 2008/Accepted 26 November 2008
Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T
cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal
inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after
reconstitution with CD4?CD45RBhighor CD4?CD45RBhighplus CD4?CD45RBlowT cells. Mice were sacrificed
at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to
produce any clinical effect on SCID mice reconstituted with CD4?CD45RBhighand CD4?CD45RBlowT cells, but
The latter was associated with an increase in the number of CD4?V?8?T cells expressing CD69 and a significantly
lower number of CD4?CD25?Foxp3?T cells. These changes were not observed in SCID mice reconstituted with
feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that
Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.
Inflammatory bowel disease (IBD) is a chronic inflammatory
condition associated with alteration of immunoregulatory
mechanisms responsible for the control of immune responses
to commensal microbiotas and their products (15, 34). Under
normal conditions, commensal microflora influences the devel-
opment and function of local and systemic immune responses
limiting an overactive inflammatory response (23, 38, 44). Con-
versely, bacterial pathogens or their products can stimulate
both innate and acquired immune responses, resulting in overt
acute and chronic mucosal inflammation (22).
Superantigens (SAgs) are microbial proteins that activate
large subsets of T or B lymphocytes. Staphylococcal enterotox-
ins, toxic shock syndrome toxin 1, streptococcal SAg, and My-
coplasma arthritidis mitogen are examples of T-cell SAgs (24,
26, 28). T-cell SAgs bind to the variable region of the T-cell
receptor (TCR) ? or ? chain and cross-link with the major
histocompatibility complex class II molecules (11, 13, 18, 29).
Oral administration of Staphylococcus aureus enterotoxin B
(SEB) induces a transient mucosal T-cell activation followed
by persistent anergy and deletion of T cells bearing the SEB-
reactive V?8 TCR for up to 4 weeks after the treatment (33,
47). Given the large number of SAg-producing microbial
agents in the gut flora, it is probable that the mechanism
involved in regulation of mucosal immune T-cell responses to
microbial SAgs is critical to the prevention of commensal bac-
terium-induced chronic inflammation (32). Furthermore, SAgs
have been implicated in immune-mediated diseases such as
rheumatoid arthritis, multiple sclerosis, psoriasis, and IBD (25,
26, 39, 40, 50, 51). Skewed TCR repertoires have been identi-
fied in patients with IBD (5, 37, 46), and a SAg-like protein
derived from Pseudomonas fluorescens, I2, was identified in
colonic lesions of over 50% of Crohn’s disease patients in a
study (8, 10, 14). However, the exact mechanism defining how
SAg may contribute to inflammation in the intestinal mucosa is
Here we investigated the role of regulatory T (Treg) cells in
the effect of orally administered SEB on T-cell subsets and on
the development of mucosal inflammation. SCID mice were
CD45RBhighT cells alone or CD4?CD45RBhighT cells to-
gether with CD4?CD45RBlowT cells. While feeding SEB had
no clinical effect on SCID mice reconstituted with both CD4?
CD45RBhighand CD4?CD45RBlowT cells, feeding SEB ac-
celerated the development of colitis in SCID mice reconsti-
tuted with CD4?CD45RBhighT cells alone. This was associ-
ated with activation and expansion of SEB-reactive CD4?
V?8?T cells and prevention of the development of T cells
expressing Foxp3. These results suggest that Treg cells modu-
late effector T-cell responses to enteric bacterium-derived
SAgs, preventing excessive activation of mucosal T cells and
preserving the normal intestinal structure and function.
MATERIALS AND METHODS
Mice. Congenic C.B-17 SCID mice and BALB/c mice were obtained from
Harlan (Indianapolis, IN). DO11.10 breeders were purchased from Charles
River Laboratories (Wilmington, MA). Female mice between 8 and 12 weeks of
* Corresponding author. Mailing address: Division of Gastroenter-
ology, Mount Sinai Hospital, Room 431, 600 University Avenue, To-
ronto, Ontario M5G 1X5, Canada. Phone: (416) 586-4800, ext. 7454.
Fax: (416) 586-4747. E-mail: KCroitoru@mtsinai.on.ca.
?Published ahead of print on 8 December 2008.
age were used in these studies. All animal experiments were performed in
accordance with institutional guidelines as approved by the Animal Care Review
Board of McMaster University. All mice were housed under specific-pathogen-
free conditions at the central animal facility at McMaster University. Donor and
recipient mice in our colony were routinely screened for Helicobacter species
infection by PCR capable of detecting ribosomal sequences common to all
Helicobacter species and were free of infection (48).
Isolation and purification of CD45RBhighand CD45RBlowCD4?spleen cells.
CD4?T-cell subsets from the spleens of BALB/c and DO11.10 mice were
isolated and sorted as described previously (36). Briefly, single-cell suspensions
were depleted of B220?, MAC1?, and CD8?cells by negative selection using
M-450 sheep anti-rat immunoglobulin G-coated Dynabeads (Dynal Biotech,
Oslo, Norway). Purified anti-CD8?, anti-CD11b, and anti-MAC1 antibodies
were obtained from BD PharMingen (Mississauga, Ontario, Canada). CD4?
CD45RBhighand CD4?CD45RBlowT-cell fractions were sorted on a FACS-
Vantage SE cell sorter (BD Biosciences, San Jose, CA) under sterile conditions.
The purity of each subpopulation was ?98%.
Reconstitution of SCID-bg mice with T-cell subsets and SEB treatment.
BALB/c- and DO11.10-derived CD4?CD45RBhighand CD4?CD45RBlowT
cells were washed and resuspended at 2 ? 106cells/ml in sterile phosphate-
buffered saline (PBS). Eight- to 12-week-old SCID mice each received either
CD4?CD45RBhighT cells (4 ? 105cells/mouse, intraperitoneally) alone or
combined with CD45RBlowCD4?T cells (2 ? 105cells/mouse) from BALB/c
mice or CD4?CD45RBhighand CD45RBlowCD4?T cells from BALB/c and
DO11.10 mice or DO11.10 and BALB/c mice, respectively. At 3 and 7 days after
T-cell reconstitution and before the onset of colitis in SCID mice that received
CD4?CD45RBhighcells, recipient SCID mice were fed 10 ?g of SEB (Sigma, St.
Louis, MO) by gavage (intragastrically) in 200 ?l of PBS with 400 ?g of soybean
trypsin inhibitor (Sigma) or soybean trypsin inhibitor alone in PBS. Mice were
euthanized at different points of time after the second administration of SEB.
Histological examination. To determine if there was a difference in or an effect
on the development of chronic colonic inflammation, which usually takes about 6 to
8 weeks, mice were euthanized at the moment at which most animals receiving SEB
reached end point or maximum weight loss (i.e., 6 weeks). At the time of harvesting,
the colon was opened longitudinally and separated into ascending, transverse, and
descending colon and cecum. Tissues were fixed in 10% buffered formalin and
sectioned and stained with hematoxylin and eosin. Each segment was analyzed for
the severity of intestinal inflammation and graded by a gastrointestinal pathologist
(C.J.S.) on a scale from 0 (no change) to 4 (most severe), as described previously
(20). The scores at each segment were combined to provide an overall score of
inflammation with a maximum score of 16.
LPL isolation. Lamina propria lymphocytes (LPL) were prepared as previ-
ously described (9). Briefly, the small intestines from a group of four to five mice
were removed and the Peyer’s patches were carefully excised. For removal of
epithelial cells and intraepithelial lymphocytes, the intestines were washed and
cut into small pieces, and then the pieces were incubated with calcium- and
magnesium-free Hanks’ balanced salt solution supplemented with 10% 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 10% bovine calf serum, antibiotics, 25 mM HEPES,
and 1.5 mg/ml collagenase A (Roche Diagnostics, Indianapolis, IN) for 30 min at
37°C with stirring. The digestion was repeated three times. The isolated cells
were pooled and separated on a 40/75% discontinuous Percoll gradient (Phar-
macia, Piscataway, NJ) centrifuged at 600 ? g and 25°C for 20 min.
Phenotypic analysis by flow cytometry. For flow cytometry analysis, suspen-
sions of 5 ? 105mononuclear cells were suspended in PBS-0.2% (wt/vol) bovine
serum albumin supplemented with 0.1% (wt/vol) sodium azide and then incu-
bated with relevant monoclonal antibody for 30 min at 4°C and washed. Three-
or four-color flow cytometry acquisition was performed on a FACScan sorter
(BD Biosciences). The following reagents and antibodies were obtained from BD
PharMingen: fluorescein isothiocyanate-conjugated hamster anti-CD3ε (145-2C-
11), phycoerythrin (PE)- and CyChrome-conjugated anti-CD4 monoclonal anti-
body (L3T4), PE-conjugated CD25 (interleukin-2 [IL-2] receptor ? chain, p55),
and PE- and fluorescein isothiocyanate-conjugated anti-F23.1 (V?8.1-3). Alexa
Fluor (488)-conjugated anti-mouse Foxp3 and allophycocyanin-conjugated anti-
CD4 antibody were obtained from Biolegend (San Diego, CA), and PE-Cy5.5-
conjugated anti-mouse DO11.10 TCR (KJ1-26) antibody was from eBioscience
(San Diego, CA). A total of 5 ? 105events gated on lymphocytes were collected
by a FACScan sorter using the CellQuest software, and the data were analyzed
with WinList version 5.0 (Verity Software House, Topsham, ME).
T-cell proliferation assays. For the T-cell proliferation assay, 5 ? 105spleno-
cytes were added to 96-well flat-bottomed tissue culture plates in Dulbecco
modified Eagle medium supplemented with 10% heat-inactivated fetal bovine
serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 50
?M 2-mercaptoethanol (Sigma-Aldrich) and stimulated with SEB (5 ?g/ml) for
72 h at 37°C in 5% CO2. The SAg SEB (5 ?g/ml) was used as a stimulator. Cell
cultures were pulsed with 1 ?Ci of [3H]thymidine for the last 16 h, and prolif-
erative responses were determined by measuring [3H]thymidine incorporation.
Statistical analysis. Data were expressed as means ? standard errors of the
means (SEM). Statistical analysis was performed using the two-tailed Student t
test for independent samples. The Mann-Whitney test was used for nonpara-
metric data. One-way analysis of variance (ANOVA) was used for time course
data. The differences between the means of two groups were considered signif-
icant when the value of P was ?0.05.
Feeding SEB to SCID mice reconstituted with CD4?
CD45RBhighT cells accelerated onset of colitis. Feeding SEB
causes rapid activation and cytokine production by T cells in
murine gut-associated lymphoid tissues (47). To examine
whether feeding SEB can influence the development of intes-
tinal inflammation, we fed SEB to SCID mice during the first
CD45RBhighT cells. Control PBS-fed SCID mice reconsti-
tuted with CD4?CD45RBhighT cells developed a gradual and
persistent weight loss with signs of diarrhea starting around 4
to 5 weeks after cell transfer. These mice developed bloody
diarrhea and rectal prolapse by 8 to 10 weeks after reconsti-
tution. The histology of the colon in these mice showed signif-
icant epithelial cell hyperplasia, lymphocyte infiltration, goblet
cell depletion, and the occasional ulceration and crypt abscess
as previously described (27). In contrast, SCID mice reconsti-
tuted with BALB/c CD4?CD45RBhighT cells and fed SEB at
days 3 and 7 after reconstitution developed significant weight
loss beginning 24 h after the second feeding. The majority of
these mice lost 20% of their original body weight within 4 to 6
weeks (Fig. 1A). The differences in body weight gain between
SEB-fed and PBS-fed CD4?CD45RBhighT-cell recipients
were statistically significant (P ? 0.05 by ANOVA).
Microscopic examination of the colon showed a significant
amplification of colonic inflammation in SEB-fed mice with
extensive epithelial hyperplasia, massive lymphocyte infiltra-
tion, and numerous crypt abscesses (Fig. 2). Overall histolog-
ical evaluation of all the segments of the colon showed that
feeding SEB was associated with a significant increase in the
severity of colitis. The mean histological score was 6.5 ? 1.0 for
PBS-fed SCID mice reconstituted with CD4?CD45RBhighT
cells, whereas the average score for SEB-fed SCID recipients
was 11.0 ? 2.0 (Fig. 1B, P ? 0.01). Thus, in the absence of Treg
cells, feeding SEB to CD45RBhighT-cell-reconstituted SCID
mice caused a more severe colitis.
SEB feeding has no clinical effect on SCID mice reconstituted
with both CD4?CD45RBhighand CD4?CD45RBlowT cells.
SCID mice reconstituted with CD4?CD45RBhighand CD4?
CD45RBlowT cells do not develop colitis (41). Feeding SEB to
SCID mice that received both CD4?CD45RBhighand CD4?
CD45RBlowT cells also failed to induce weight loss (Fig. 1A)
or histological evidence of inflammation (Fig. 1B). Similar
results were observed in SCID mice reconstituted with unsepa-
rated CD4?T cells (data not shown). In fact, repeated oral
administration of SEB (10 ?g per mouse, twice a week for 4
weeks) to SCID mice reconstituted with combined CD4?
CD45RBhighand CD4?CD45RBlowT cells failed to cause
weight lose or colitis (data not shown).
708HERIAZON ET AL.INFECT. IMMUN.
Feeding SEB to SCID mice reconstituted with CD4?
CD45RBhighT cells leads to early activation of T cells in the
absence of Treg cells. The significant weight loss and clinical
signs of colitis seen immediately after the second SEB feeding in
SCID mice reconstituted with CD4?CD45RBhighT cells sug-
gested that oral SEB caused early T-cell activation. We examined
the induction of T-cell early-activation marker CD69 on CD4?
V?8?T cells in the spleen, mesenteric lymph nodes (MLN), and
LPL from SCID mice reconstituted with CD4?CD45RBhighT
cells during the first 72 h after the second SEB feeding. In SEB-
fed BALB/c mice, a significant increase in the expression of
CD69?was detected on T cells in the LPL and a small but not
statistically significant increase was detected in the MLN (Fig.
3A). On the other hand, feeding SEB to SCID mice reconstituted
with CD4?CD45RBhighT cells alone caused a significant (P ?
0.05) increase in the proportion of CD4?V?8?T cells expressing
CD69 in LPL and MLN (Fig. 3B). There was no evidence of
increased expression of CD69 on CD4?V?8?T cells in any of
the tissues from SCID mice reconstituted with CD45RBhighT
cells and fed PBS alone, in spite of the development of colitis.
Feeding SEB induced expansion of CD4?V?8?T cells in
SCID mice reconstituted with CD4?CD45RBhighT cells. Pre-
vious studies showed that oral administration of SEB at high
doses (e.g., 50 to 200 ?g/mouse) or repeated oral administration
of SEB at low doses (e.g., 1 to 10 ?g/mouse) to immunocompe-
recovered within 4 weeks (33, 36, 47). We examined the changes
in CD4?V?8?T cells in both mucosal and peripheral tissues at
different time points after feeding SEB. We found that feeding
SEB to SCID mice reconstituted with CD4?CD45RBhighT cells
alone led to an increase in the percentage of CD4?V?8?T cells
in spleen, MLN, and LPL by 12 h after the second feeding, and
this remained significantly elevated for at least 4 weeks, while the
percentage of CD4?V?8?T cells in the PBS-fed SCID recipi-
ents was unchanged (Fig. 4).
CD45RBlowT cells limited CD45RBhighT-cell expansion in
reconstituted SCID mice regardless of SEB administration.
To determine if SEB induced a chronic effect on expansion of
RBhigh-derived T cells and if RBlowwas effective at controlling
expansion of RBhighT cells in SCID mice receiving both types
(2), we examined the effect of SEB on the expansion of
CD45RBhighT cells in vivo, in the presence or absence of
CD45RBlowTreg cells after 7 weeks. SCID mice were recon-
CD45RBlowT-cell subsets from DO11.10 and BALB/c donor
mice. CD4?T cells from DO11.10 mice possess the transgenic
TCR (KJ1-26?) specific for ovalbumin. This TCR has the V?8
chain that is reactive with SEB (52) and can be differentiated
from the BALB/c TCR by the clonotype-specific monoclonal
antibody KJ1-26. The effect of SEB on CD4?CD45RBhighT
cells from BALB/c (BALB/c RBhigh) donors transferred into
SCID mice was compared with the effect in mice receiving both
BALB/c RBhighdonor cells plus DO11.10 CD4?CD45RBlow
donor cells (DO11.10 RBlow) or DO11.10 RBhighdonor cells
combined with BALB/c RBlowdonor cells (Fig. 5). SCID re-
cipient mice were fed SEB or PBS at 3 and 7 days after T-cell
reconstitution, and the number of CD4?T cells was estimated
using the total number of mononuclear cell counts obtained
after collection of spleen and the percentage of CD4?cells
detected in flow cytometry analysis. SEB did not have a chronic
effect on total CD4?T-cell expansion in spleen or MLN from
SCID mice reconstituted with RBhighcells alone or RBhighand
RBlowcells as observed at 7 weeks posttransfer. Furthermore,
RBlowcells significantly limited expansion of RBhighT cells in
FIG. 2. Representative photomicrographs of transverse colon from
SCID mice 6 weeks after reconstitution with purified CD4?
CD45RBhighT-cell subset. Marked increases in epithelial hyperplasia,
lymphocyte infiltration, and crypt abscesses were seen in SEB-fed
recipients (B) compared with PBS-fed recipients (A). Hematoxylin and
eosin stain. Original magnification, ?100.
FIG. 1. Oral SEB activates colitis in SCID mice reconstituted with
CD4?CD45RBhighT cells. (A) Eight- to 12-week-old female C.B-17
SCID mice were reconstituted with 4 ? 105CD4?CD45RBhighT cells or
4 ? 105CD4?CD45RBhighplus 2 ? 105CD4?CD45RBlowT cells
at days 3 and 7. The changes in weight over time are expressed as per-
centages of initial body weight. The differences in body weight between
SEB-fed and PBS-fed CD4?CD45RBhighT-cell recipients were statisti-
cally significant (P ? 0.05 by ANOVA). SEB feeding had no significant
effect on SCID mice reconstituted with both CD4?CD45RBhighT cells
and CD4?CD45RBlowT cells. Data are representative of three indepen-
dent experiments with four to five mice in each group. (B) Histological
scores of colitis in SCID mice reconstituted with CD4?T-cell subsets
colonic tissues were collected for histology examination. Inflammation
was scored for the cecum and the proximal, middle, and distal colon, as
described in Materials and Methods. Each data point represents the score
of an individual mouse. The bars represents the means of the inflamma-
tory scores for the groups. P ? 0.01, SEB-fed versus PBS-fed SCID mice
reconstituted with CD4?CD45RBhighT cells.
VOL. 77, 2009Treg MODULATION OF SEB-ACCELERATED COLITIS709
both SEB- and PBS-fed SCID mice receiving both cell types
compared with SCID mice receiving only RBhighcells.
Feeding SEB impaired CD4?CD25?Foxp3?T-cell devel-
opment in SCID mice reconstituted with CD4?CD45RBhighT
cells alone. In order to determine if the presence of Treg cells
modulates the development or function of effector T cells in
mice fed SEB, we examined the expression of Treg cell mark-
ers in mice receiving donor cells. Feeding SEB significantly
decreased the percentages of CD25?CD4?, Foxp3?CD4?,
and CD25?Foxp3?CD4?T cells in spleens of SCID mice that
received BALB/c CD4?CD45RBhighT cells alone (P ? 0.05),
suggesting that SEB prevented or delayed development of
CD45RBhigh-derived Treg cells (Fig. 6; only CD25?Foxp3?
double-positive CD4?T cells are shown). However, SEB failed
to alter the percentages of T cells expressing these Treg mark-
ers in spleens from SCID mice reconstituted with both RBhigh
and RBlowT cells, indicating that Treg RBlowT cells modu-
lated the effect of SEB on RBhigh-derived Treg cells. More-
over, SEB did not affect RBlow-derived Treg cells (data not
The results presented here demonstrated that in the absence of
Treg cells, mucosal exposure to a bacterially derived product with
FIG. 3. Activation of mucosal T cells after SEB feeding of SCID mice
reconstituted with CD45RBhigh
T cells. Naïve BALB/c (A) or
CD45RBhighT-cell-reconstituted SCID (B) mice were fed SEB or PBS
twice as described. Lymphocytes from the spleen (SPL), MLN, or lamina
propria were harvested 12 h after the last feeding and stained for three-
color flow cytometric analysis. The expression of the activation marker
CD69 was analyzed on the gated CD4?V?8?T-cell subpopulation. The
results are expressed as the mean percentage for each group ? SEM. The
results shown are representative of two independent experiments with
seven to eight mice in each group.*, P ? 0.05 versus PBS-fed group.
FIG. 4. Feeding SEB to SCID mice reconstituted with BALB/c CD4?
reconstituted with CD4?CD45RBhighT cells were fed SEB or PBS, as
described. The percentages of CD4?V?8?T cells in total CD4?cell
populations in the spleen (SPL), MLN, and lamina propria were deter-
mined at various time points as indicated after cell reconstitution. CD4?
V?8?T cells from all the examined compartments remained stable in
PBS-fed SCID recipients. In contrast, SEB-reactive CD4?V?8?T cells
increased shortly after SEB feeding and remained significantly elevated
up to 28 days after transfer before dropping to the same level as that in
PBS-fed mice. Data shown are means ? SEM, with n ? 3 to 5 for each
710HERIAZON ET AL.INFECT. IMMUN.
SAg activity, i.e., SEB, significantly activated the development of
chronic intestinal inflammation. Specifically, oral administration
of SEB to SCID mice reconstituted with CD4?CD45RBhighT
cells was associated with activation of effector CD4?T cells and
expansion of SEB-reactive V?8?T cells. Therefore, in the ab-
sence of Treg cells, bacterially derived SAg induced activation
and expansion of effector T cells that may have accelerated the
onset of colitis. In addition, oral administration of SEB to SCID
mice reconstituted with CD4?CD45RBhighT cells impaired de-
velopment of T cells expressing the Treg phenotypes, i.e., T cells
Previous studies suggested that SAg-induced mucosal T-cell
stimulation may be implicated in the development of IBD as
evidenced by skewed TCR V? usage in IBD patients (1, 20).
Indeed, systemic administration of the bacterial SAg SEB to mice
induces a self-limiting enteropathy (6, 32) with stimulation of
CD4?and CD8?T cells (19, 43) and release of tumor necrosis
factor alpha, IL-1, IL-2, IL-6, and gamma interferon (26, 28). SAg
healthy and inflamed colonic mucosa, suggesting that SAg could
be an important initiator of the inflammatory cascade through
direct T-cell activation (12). Therefore, our finding that feeding
SEB accelerated and aggravated colitis in SCID mice reconsti-
tuted with RBhighT cells alone and that oral SEB induced expan-
sion of responsive CD4?V?8?T cells in SCID mice reconsti-
which mucosal exposure to a SAg can induce mucosal inflamma-
Under normal circumstances interactions between the intesti-
nal microflora and the host immune system are tightly regulated,
preventing excessive local inflammation (15, 34). Feeding SAg to
immunocompetent mice with normal gut flora fails to induce
mucosal inflammation, suggesting that the normal mucosal envi-
ronment also prevents or modulates the immune response to
luminal SAg exposure (16, 31, 35). In addition, direct mucosal
administration, e.g., intrarectal administration, of SEB to normal
immunocompetent mice failed to induce inflammation (30).
These findings support our results showing that feeding SEB to
SCID mice reconstituted with combined CD4?CD45RBhighand
CD4?CD45RBlowT cells failed to induce T-cell activation or
CD4?V?8?T-cell expansion. Furthermore, oral administration
of SEB in naïve BALB/c mice and SCID mice reconstituted with
both CD4?CD45RBhighand CD4?CD45RBlowT cells, or un-
separated CD4?T cells, failed to induce any significant intestinal
damage. Therefore, mucosal exposure to a SAg in the intestine is
not sufficient to cause mucosal inflammation.
Several immune mechanisms, including Treg cells, restrict
and regulate the response of the mucosal immune system to
mucosal bacterial antigens (16, 35). An alteration of immune
regulatory mechanisms in response to microbial products may
result in the development of chronic inflammatory diseases
such as Crohn’s disease and ulcerative colitis (7, 49). Our
results showed that SEB impaired development of RBhigh-
derived Treg cells. However, SEB did not affect Treg cells from
the RBlowsubset or RBhigh-derived Treg cells in the presence
of RBlow. This would suggest that in addition to direct T-cell
FIG. 5. CD45RBlowT cells limited CD45RBhighT-cell expansion in reconstituted SCID mice regardless of SEB administration. Eight- to 12-week-old
female C.B-17 SCID mice were reconstituted with 4 ? 105CD4?CD45RBhighT cells derived from BALB/c (BALB/c RBhighsingle-transfer) mice or
4 ? 105CD4?CD45RBhighplus 2 ? 105CD4?CD45RBlowT cells derived from BALB/c and DO11.10 (BALB/c RBhighdouble-transfer) mice,
respectively, or DO11.10 and BALB/c (DO11.10 RBhighdouble-transfer) mice, respectively. Data shown are means of RBhigh-derived T cells ? SEM,
n ? 3, and significance was determined at P ? 0.05. Lines above error bars indicate significant differences between recipient models within control or
SEB-treated mice. No significant differences were observed between SEB-treated mice and PBS-treated controls.
VOL. 77, 2009Treg MODULATION OF SEB-ACCELERATED COLITIS711
activation, SEB may alter development and function of RBhigh-
derived Treg cells and that this effect is also modulated by the
presence of Treg cells. It is not known how SEB affects the
development of Treg cells, but it is possible that SEB altered
the conversion of naïve T cells into effector T cells, while
somehow blocking the expression of Foxp3 and the develop-
ment of Treg cells. However, it is also likely that the microen-
vironment (e.g., cytokine and chemokine milieu) promoted by
the conversion of naïve T cells into effector T cells might not
have allowed for the development of Treg cells (45).
Finally, RBhighT-cell proliferation or expansion in vivo was
limited by the presence of RBlowT cells. In SCID mice recon-
stituted with CD4?CD45RBhighT cells, the number of CD4?
T cells recovered from spleen lymphocytes and MLN at the
time of colitis was three to six times higher than that found in
SCID mice reconstituted with both CD4?CD45RBhighand
CD4?CD45RBlowT-cell subsets (20). Previous studies
showed that administration of regulatory cytokines (e.g., IL-
10) protected recipient SCID mice reconstituted with CD4?
CD45RBhighT cells from disease and decreased the number of
recovered splenic CD4?T cells (42). This may partially explain
how Treg cells regulated RBhighT-cell expansion in cotransfer
to SCID mice. The finding that germfree mice do not develop
oral tolerance to a fed antigen has led to the proposition that
commensal microflora is important for the development of
Treg cell function involved in the acquisition of tolerance (21).
The mechanisms regulating the expansion and survival of
CD4?T cells in a mouse with a normal gut flora may therefore
also involve Treg cells (2, 3, 17). Since most animal models of
colitis like SCID mice reconstituted with naïve CD4?
CD45RBhighT cells are dependent on the presence of com-
mensal flora (4, 49), the CD4?T-cell expansion and infiltration
into mucosal sites are likely a reflection of bacterial activation.
All together, the observation that mucosal exposure to bacte-
rial SAg activated the development of intestinal inflammation in
immunodeficient hosts may explain how gut flora and bacterium-
immune-dysregulated intestinal mucosa. The current study also
demonstrated that Treg cells are critical to the control of T-cell
responses to luminal bacterium-derived products and prevent po-
tentially damaging inflammatory responses. In addition, we have
demonstrated how SEB can alter Treg development, which con-
tributes to the activation of effector T cells in a dysregulated
environment. Therefore, our findings indicated that in the ab-
sence of Treg cells, a dysregulated response to SAg could lead to
T-cell activation that synergizes with commensal gut flora to ini-
tiate and aggravate colitis.
This work was funded by a grant from the Canadian Institute of Health
Research (MOP#74470). Ken Croitoru has received support as an On-
tario Minister of Health Career Scientist and as a Crohn’s and Colitis
Foundation of Canada IBD Scientist. Pengfei Zhou held a scholarship
from the Natural Sciences and Engineering Research Council of Canada.
FIG. 6. Oral SEB impaired development of CD4?CD25?Foxp3?T cells in the absence of CD4?CD45RBlowT cells. Eight- to 12-week-old
female C.B-17 SCID mice were reconstituted with 4 ? 105CD4?CD45RBhighT cells derived from BALB/c (BALB/c RBhigh) mice or 4 ? 105
CD4?CD45RBhighplus 2 ? 105CD4?CD45RBlowT cells derived from BALB/c and DO11.10 (BALB/c RBhigh? DO11.10 RBlow) mice,
respectively, or DO11.10 and BALB/c (DO11.10 RBhigh? BALB/c RBlow) mice, respectively. Mice were fed SEB (10 ?g/mouse) or PBS at days
3 and 7. Data shown are means of RBhigh-derived T cells ? SEM, n ? 3, and significance was determined (P ? 0.05).
712HERIAZON ET AL.INFECT. IMMUN.
1. Aisenberg, J., E. C. Ebert, and L. Mayer. 1993. T-cell activation in human
intestinal mucosa: the role of superantigens. Gastroenterology 105:1421–1430.
2. Annacker, O., O. Burlen-Defranoux, R. Pimenta-Araujo, A. Cumano, and A.
Bandeira. 2000. Regulatory CD4 T cells control the size of the peripheral
activated/memory CD4 T cell compartment. J. Immunol. 164:3573–3580.
3. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A.
Cumano, and A. Bandeira. 2001. CD25? CD4? T cells regulate the expan-
sion of peripheral CD4 T cells through the production of IL-10. J. Immunol.
4. Aranda, R., B. C. Sydora, P. L. McAllister, S. W. Binder, H. Y. Yang, S. R.
Targan, and M. Kronenberg. 1997. Analysis of intestinal lymphocytes in
mouse colitis mediated by transfer of CD4?, CD45RBhigh T cells to SCID
recipients. J. Immunol. 158:3464–3473.
5. Baca-Estrada, M. E., D. K. Wong, and K. Croitoru. 1995. Cytotoxic activity
of V beta 8? T cells in Crohn’s disease: the role of bacterial superantigens.
Clin. Exp. Immunol. 99:398–403.
6. Benjamin, M. A., J. Lu, G. Donnelly, P. Dureja, and D. M. McKay. 1998.
Changes in murine jejunal morphology evoked by the bacterial superantigen
Staphylococcus aureus enterotoxin B are mediated by CD4?T cells. Infect.
7. Bouma, G., and W. Strober. 2003. The immunological and genetic basis of
inflammatory bowel disease. Nat. Rev. Immunol. 3:521–533.
8. Chiba, M., T. Nakamura, S. Hoshina, and Y. Kitagawa. 2001. Optimal cases
and sites to search for primary microbial agents in Crohn’s disease. Gastro-
9. Cong, Y., S. L. Brandwein, R. P. McCabe, A. Lazenby, E. H. Birkenmeier, J. P.
Sundberg, and 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. 187:855–864.
10. Dalwadi, H., B. Wei, M. Kronenberg, C. L. Sutton, and J. Braun. 2001. The
Crohn’s disease-associated bacterial protein I2 is a novel enteric T cell
superantigen. Immunity 15:149–158.
11. Dellabona, P., J. Peccoud, J. Kappler, P. Marrack, C. Benoist, and D.
Mathis. 1990. Superantigens interact with MHC class II molecules outside of
the antigen groove. Cell 62:1115–1121.
12. Dionne, S., S. Laberge, C. Deslandres, and E. G. Seidman. 2003. Modulation
of cytokine release from colonic explants by bacterial antigens in inflamma-
tory bowel disease. Clin. Exp. Immunol. 133:108–114.
13. Drake, C. G., and B. L. Kotzin. 1992. Superantigens: biology, immunology
and potential role in disease. J. Clin. Immunol. 12:149–162.
14. Elson, C. O. 2000. Commensal bacteria as targets in Crohn’s disease. Gas-
15. Elson, C. O., and Y. Cong. 2002. Understanding immune-microbial ho-
meostasis in intestine. Immunol. Res. 26:87–94.
16. Fagarasan, S., and T. Honjo. 2003. Intestinal IgA synthesis: regulation of
front-line body defences. Nat. Rev. Immunol. 3:63–72.
17. Freitas, A. A., and B. Rocha. 2000. Population biology of lymphocytes: the
flight for survival. Annu. Rev. Immunol. 18:83–111.
18. Herman, A., J. W. Kappler, P. Marrack, and A. M. Pullen. 1991. Superan-
tigens: mechanism of T-cell stimulation and role in immune responses.
Annu. Rev. Immunol. 9:745–772.
19. Herrmann, T., and H. R. MacDonald. 1993. The CD8 T cell response to
staphylococcal enterotoxins. Semin. Immunol. 5:33–39.
20. Ibbotson, J. P., and J. R. Lowes. 1995. Potential role of superantigen induced
activation of cell mediated immune mechanisms in the pathogenesis of
Crohn’s disease. Gut 36:1–4.
21. Jiang, H. Q., M. C. Thurnheer, A. W. Zuercher, N. V. Boiko, N. A. Bos, and
J. J. Cebra. 2004. Interactions of commensal gut microbes with subsets of
B- and T-cells in the murine host. Vaccine 22:805–811.
22. Kaufmann, S. H., and U. E. Schaible. 2005. Antigen presentation and rec-
ognition in bacterial infections. Curr. Opin. Immunol. 17:79–87.
23. Kelly, D., S. Conway, and R. Aminov. 2005. Commensal gut bacteria: mech-
anisms of immune modulation. Trends Immunol. 26:326–333.
24. Kilpatrick, D. C. 1999. Mechanisms and assessment of lectin-mediated mi-
togenesis. Mol. Biotechnol. 11:55–65.
25. Kotzin, B. L., D. Y. Leung, J. Kappler, and P. Marrack. 1993. Superantigens
and their potential role in human disease. Adv. Immunol. 54:99–166.
26. Lavoie, P. M., J. Thibodeau, F. Erard, and R.-P. Sekaly. 1999. Understand-
ing the mechanism of action of bacterial superantigens from a decade of
research. Immunol. Rev. 168:257–269.
27. Leach, M. W., A. G. Bean, S. Mauze, R. L. Coffman, and F. Powrie. 1996.
Inflammatory bowel disease in C.B-17 scid mice reconstituted with the
CD45RBhigh subset of CD4? T cells. Am. J. Pathol. 148:1503–1515.
28. Li, H., A. Llera, E. L. Malchiodi, and R. A. Mariuzza. 1999. The structural
basis of T cell activation by superantigens. Annu. Rev. Immunol. 17:435–466.
29. Licastro, F., L. J. Davis, and M. Morini. 1993. Lectins and superantigens:
membrane interactions of these compounds with T lymphocytes affect im-
mune responses. Int. J. Biochem. 25:845–852.
30. Lu, J., A. Wang, S. Ansari, R. M. Hershberg, and D. M. McKay. 2003. Colonic
bacterial superantigens evoke an inflammatory response and exaggerate disease
in mice recovering from colitis. Gastroenterology 125:1785–1795.
31. Macpherson, A. J., and T. Uhr. 2004. Induction of protective IgA by intes-
tinal dendritic cells carrying commensal bacteria. Science 303:1662–1665.
32. McKay, D. M. 2001. Bacterial superantigens: provocateurs of gut dysfunction
and inflammation? Trends Immunol. 22:497–501.
33. Migita, K., and A. Ochi. 1994. Induction of clonal anergy by oral adminis-
tration of staphylococcal enterotoxin B. Eur. J. Immunol. 24:2081–2086.
34. Nagler-Anderson, C. 2001. Man the barrier! Strategic defences in the intes-
tinal mucosa. Nat. Rev. Immunol. 1:59–67.
35. Nagler-Anderson, C., A. K. Bhan, D. K. Podolsky, and C. Terhorst. 2004.
Control freaks: immune regulatory cells. Nat. Immunol. 5:119–122.
36. Nishimura, M., Y. Fujiyama, M. Niwakawa, T. Sasaki, and T. Bamba. 2002.
In vivo cytokine responses in gut-associated lymphoid tissue (GALT) and
spleen following oral administration of staphylococcal enterotoxin B. Immu-
nol. Lett. 81:77–85.
37. Ogawa, H., H. Ito, A. Takeda, S. Kanazawa, M. Yamamoto, H. Nakamura, Y.
Kimura, K. Yoshizaki, and T. Kishimoto. 1997. Universal skew of T cell
receptor (TCR) V beta usage for Crohn’s disease (CrD). Biochem. Biophys.
Res. Commun. 240:545–551.
38. Ouwehand, A., E. Isolauri, and S. Salminen. 2002. The role of the intestinal
microflora for the development of the immune system in early childhood.
Eur. J. Nutr. 41(Suppl. 1):I32–I37.
39. Paliard, X., S. G. West, J. A. Lafferty, J. R. Clements, J. W. Kappler, P.
Marrack, and B. L. Kotzin. 1991. Evidence for the effects of a superantigen
in rheumatoid arthritis. Science 253:325–329.
40. Posnett, D. N., I. Schmelkin, D. A. Burton, A. August, H. McGrath, and L. F.
Mayer. 1990. T cell antigen receptor V gene usage. Increases in V beta 8?
T cells in Crohn’s disease. J. Clin. Investig. 85:1770–1776.
41. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, and 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. 5:1461–1471.
42. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, and R. L. Coffman.
1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid
mice reconstituted with CD45RBhi CD4? T cells. Immunity 1:553–562.
43. Quaratino, S., G. Murison, R. E. Knyba, A. Verhoef, and M. Londei. 1991.
Human CD4?CD8????T cells express a functional T cell receptor and can
be activated by superantigens. J. Immunol. 147:3319–3323.
44. Rakoff-Nahoum, S., and R. Medzhitov. 2006. Role of the innate immune
system and host-commensal mutualism. Curr. Top. Microbiol. Immunol.
45. Samanta, A., B. Li, X. Song, K. Bembas, G. Zhang, M. Katsumata, S. J.
Saouaf, Q. Wang, W. W. Hancock, Y. Shen, and M. I. Greene. 2008. TGF-
beta and IL-6 signals modulate chromatin binding and promoter occupancy
by acetylated FOXP3. Proc. Natl. Acad. Sci. USA 105:14023–14027.
46. Saubermann, L. J., C. S. Probert, A. D. Christ, A. Chott, J. R. Turner, A. C.
Stevens, S. P. Balk, and R. S. Blumberg. 1999. Evidence of T cell receptor
beta-chain patterns in inflammatory and noninflammatory bowel disease
states. Am. J. Physiol. 276:G613–G621.
47. Spiekermann, G. M., and C. Nagler-Anderson. 1998. Oral administration of
the bacterial superantigen staphylococcal enterotoxin B induces activation
and cytokine production by T cells in murine gut-associated lymphoid tissue.
J. Immunol. 161:5825–5831.
48. Streutker, C. J., C. N. Bernstein, V. L. Chan, R. H. Riddell, and K. Croitoru.
2004. Detection of species-specific helicobacter ribosomal DNA in intestinal
biopsy samples from a population-based cohort of patients with ulcerative
colitis. J. Clin. Microbiol. 42:660–664.
49. Strober, W., I. J. Fuss, and R. S. Blumberg. 2002. The immunology of
mucosal models of inflammation. Annu. Rev. Immunol. 20:495–549.
50. Sutton, C. L., J. Kim, A. Yamane, H. Dalwadi, B. Wei, C. Landers, S. R.
Targan, and J. Braun. 2000. Identification of a novel bacterial sequence
associated with Crohn’s disease. Gastroenterology 119:23–31.
51. Torres, B. A., and H. M. Johnson. 1998. Modulation of disease by superan-
tigens. Curr. Opin. Immunol. 10:465–470.
52. Zhou, P., R. Borojevic, C. Streutker, D. Snider, H. Liang, and K. Croitoru.
2004. Expression of dual TCR on DO11.10 T cells allows for ovalbumin-
induced oral tolerance to prevent T cell-mediated colitis directed against
unrelated enteric bacterial antigens. J. Immunol. 172:1515–1523.
Editor: B. A. McCormick
VOL. 77, 2009Treg MODULATION OF SEB-ACCELERATED COLITIS 713