Endocrine self and gut non-self intersect
in the pancreatic lymph nodes
Shannon J. Turley*†, Je-Wook Lee†, Nick Dutton-Swain*, Diane Mathis*‡, and Christophe Benoist*‡
*Section on Immunology and Immunogenetics, Joslin Diabetes Center, and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical
School, One Joslin Place, Boston, MA 02215; and†Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, and Department of
Pathology, Harvard Medical School, 44 Binney Street, Boston, MA 02115
Contributed by Christophe Benoist, October 14, 2005
The autoimmune cascade that culminates in diabetes initiates
within pancreatic lymph nodes (PLNs). Here, we show that devel-
opmentally controlled lymphogenesis establishes a preferential
trafficking route from the gut to the PLN, where T cells can be
activated by antigens drained from the peritoneum and the gas-
trointestinal tract. Furthermore, intestinal stress modifies the pre-
sentation of pancreatic self-antigens in PLNs. The convergence of
endocrine and intestinal contents within PLNs has significant
implications for type 1 diabetes and may help to explain the
link between autoimmune pathogenesis and environmental
autoimmunity ? diabetes ? peritoneal lymphatics ? enteropathy
endocrine pancreas. The first stage of disease, known as insulitis,
entails leukocyte invasion of the pancreatic islets; the second
stage, overt diabetes, is marked by massive death of islet ? cells
and the subsequent loss of glucose homeostasis (1). The immune
assault on ? cells that preludes diabetes is orchestrated by T cells.
Naı ¨ve, ? cell-reactive T cells initially encounter their cognate
antigen in pancreatic lymph nodes (PLNs) (2). Islet antigens are
shuttled to these nodes by CD11b?dendritic cells and subse-
quently presented to T lymphocytes (3). Thus, PLNs are key to
diabetes pathogenesis (4, 5), the location where tolerance to
pancreatic self-antigens is first broken.
Environmental factors impinge on an individual’s genetic
susceptibility to type 1 diabetes (6). Alimentary agents such as
enteroviruses (e.g., coxsackie virus) and dietary antigens (e.g.,
gluten) are associated with T1D (7–13), and there is a frequent
association between T1D and celiac disease (14). However, the
precise mechanisms by which these factors might influence
the autoimmune response to pancreatic ? cells remain elusive.
The common entry route of such environmental components
certainly raises the question of how the gastrointestinal tract
relates to the pancreatic axis.
ype 1 diabetes (T1D) is an autoimmune disorder character-
ized by destruction of the insulin-producing ? cells of the
Materials and Methods
Mice. All mice were bred and maintained under barrier condi-
tions in the Joslin Diabetes Center animal facility in accordance
with National Institutes of Health guidelines. BDC2.5?NOD,
OT-I?Rag-10, and OT-II TCR transgenic (tg) mice are described
at the Joslin Diabetes Center animal facility, and B6 mice were
obtained from The Jackson Laboratory.
Antigens. Mice were injected i.p., intragastrically, or i.v. with
chicken ovalbumin (OVA) (Sigma), BSA (Sigma), or saline (as
controls), and OVA-coated beads, OVA-loaded apoptotic cells,
or purified pancreatic islets. Soluble OVA was adsorbed onto
0.5-?m polystyrene microparticles (beads) (Polysciences) ac-
cording to the manufacturer’s protocol. Purified pancreatic islets
were purified from 4- to 6-week-old NOD mice at the Islet Core
of the Juvenile Diabetes Research Foundation Center at Har-
vard Medical School and were resuspended in PBS for injection.
Reagents. For G?i inhibition, donor splenocytes were treated
with 100 ng?ml Pertussis toxin (Sigma) for 30 min at 37°C,
washed, and resuspended in PBS for i.p. injection. Large intes-
tine injury was performed with dextran sulfate sodium (DSS)
(MW 40,000; ICN) as described in ref. 18. DSS-containing
drinking water (2% or 5% wt?vol) was administered to the mice
for different lengths of time depending on the experiments. For
adoptive transfers, mice were given DSS water for 2 days and
then received normal drinking water for 1–3 days before T cells
were injected. Small intestine injury was induced as described in
ref. 19. Mice received a single s.c. injection of indomethacin
(INDO) (85 mg?kg) 24 h before adoptive transfers. Control
animals were fed a standard mouse diet with 20% protein,
whereas the low-protein (LP) cohort was fed an 8%-protein diet
(TestDiet) for 2–5 days before adoptive transfers and then
switched to control diet for the duration of the experiment.
adult and infant donors and processed into single-cell suspen-
sions. After red blood cell lysis, B cells and CD4?T cells were
purified by negative depletion using streptavidin-labeled Abs
and biotinylated MACS beads (Miltenyi Biotec). For B cell
selection, Abs to CD3, CD11b, and CD11c were used; for CD4?
T cell selection, Abs to B220, CD8??, CD11b, and CD11c were
used. Purified lymphocyte populations or unfractionated spleno-
cytes were then labeled with the cytoplasmic dye 5,6-carboxy-
succinimidyl-fluorescein-ester (CFSE). Fluorescently labeled
cells were then injected at different doses into the peritoneal
cavity or the bloodstream (tail vein or retroorbital sinus).
Recipients were killed no longer than 28 h after donor-cell
injections, and the relevant lymphoid organs were harvested and
processed into single-cell suspensions. After counting, the organ
suspensions were subjected to cytofluorimetric analysis, and the
fluorescently labeled cells were quantitated. Samples were ac-
quired by using Coulter instrumentation and analyzed with
Assessment of T Cell Activation. T cell proliferation was assessed by
using an adoptive transfer system with CFSE-labeled lympho-
cytes as described in ref. 2. Various lymphoid organs, including
(SLNs), and Peyer’s patches, were harvested 2–3 days after i.v.
transfer of CFSE-labeled donor T cells, and single-cell suspen-
sions were prepared by using glass-slide disruption. For cyt-
ofluorimetric analysis of T cell activation, lymph node (LN) cells
Conflict of interest statement: No conflicts declared.
Abbreviations: CFSE, 5,6-carboxy-succinimidyl-fluorescein-ester; DSS, dextran sulfate so-
dium; INDO, indomethacin; LN, lymph node; LP, low-protein; MLN, mesenteric LN; OVA,
ovalbumin; PLN, pancreatic LN; SLN, s.c. LN; T1D, type 1 diabetes; tg, transgenic.
‡To whom correspondence may be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
December 6, 2005 ?
vol. 102 ?
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were then stained with fluorescently labeled monoclonal Abs
specific for V?4 and CD4 (BDC2.5), V?2 and CD8 (OT-I), and
V?2 and CD4 (OT-II) T cells. In assessments of T cell activation,
V?4 was replaced with antibodies to CD44 or CD69. The extent
of T cell proliferation was determined simultaneously by CFSE-
Immunizations. Two days after treatment with intestinal pertur-
bants was terminated, NOD mice were anaesthetized, and a
single hind footpad was injected with 10 ?g of the BDC2.5 mimic
Insulitis Scoring. For insulitis studies, BDC2.5?NOD mice were
put on DSS-containing drinking water at weaning (21 days of
age). After 7 days, experimental and control littermates were
killed, and pancreata were promptly excised, formalin-fixed, and
embedded in paraffin. Thin sections were stained with hema-
toxylin?eosin and examined by light microscopy. Multiple non-
consecutive sections per animal were scored for insulitis (at least
50 islets per individual). The same fixation and staining proce-
dures were used for gut histology.
The experiments reported here may provide a cellular and
molecular substratum to understand how environmental factors
that enter the body via the gastrointestinal route influence
pancreas-directed autoimmunity. The first observations were
made through pure serendipity, however. For purposes of in vivo
imaging, we were attempting to transfer fluorescently labeled
splenocytes to SLNs of young NOD mice by i.p. injection. When
SLNs were harvested from recipient mice 24 h later and exam-
ined by confocal microscopy, we observed fewer cells than
expected. This was also true in MLNs and in several SLNs; the
inguinal, axillary, and brachial SLNs were examined but yielded
identical results (and will be referred to generically as SLN
throughout this study). Unexpectedly, a far larger density of
donor cells was found in PLNs, compared with SLNs or even
MLNs, which one might have expected to be a primary draining
site (Fig. 1A). Quantitation by cytofluorimetry over several such
transfers confirmed the preferential localization to PLNs of cells
infused in the peritoneum, because the density of fluorescent
cells in these nodes was 17- to 37-fold higher than in SLNs and
10- to 20-fold higher than in MLNs (Fig. 1A). The reduced
frequency of donor cells in the MLNs or SLNs after peritoneal
injection did not reflect dilution of the label provoked by some
activation event at those sites as the cells did not proliferate at
such short times after transfer (data not shown). The selective
accumulation in PLNs was not an artifact of organ size and
saturation, because PLN and SLNs are of roughly similar sizes
in normal mice, and the PLN preference was observed over a
wide range of donor-cell numbers (ranging from 2.5 to 60 ? 106
cells; data not shown).
In marked contrast, fluorescently labeled splenocytes trans-
ferred i.v. were uniformly distributed among the LNs (Fig. 1B).
Thus, there seemed to be no selective advantage in homing of
cells to PLNs or retention within them via blood vessel high
endothelial venules (HEVs). The preferential homing after i.p.
administration appeared to denote a favored lymphatic drainage
from the peritoneum to PLNs, rather than a selective retention,
which would have also been observed after homing from blood.
To define the kinetics of this unforeseen trafficking route, we
enumerated the donor cells within LNs at various time points
after i.p. infusion. Small numbers of donor cells could be
detected uniformly in LNs in the first hours, but their density
began to increase specifically in PLNs after 8 h and continued to
increase (Fig. 1C). The frequency of donor cells in MLNs and
SLNs, on the other hand, remained flat throughout the time
examined in PLNs, MLNs, inguinal LNs, and axillary LNs of 4-week-old NOD mice
24 h after i.p. injection with 10 ? 106CFSE-labeled splenocytes from 4-week-old
confocal microscopy (magnification ?250). Data are representative of 10–20
mice. The donor cell proportion among total LN cells is indicated. (B) Cytofluori-
metric enumeration of donor cells in recipient LNs after i.p. or i.v. transfer. The
cell density in SLNs (filled columns) or MLNs (open columns). Shown is data from
4–10 mice. (C) Kinetics of donor cell trafficking to PLNs, MLNs, and SLNs was
determined by cytofluorimetry. Data are shown as the number of CFSE?donor
cells (¢) ? 10?3per 1 ? 106recipient LN cells. Three or four mice were examined
donor cells (¢) ? 10?3per 1 ? 106recipient LN cells in PLNs (filled column) and
MLNs (open column). Three to four mice were used per condition. (E) Prolifera-
or OVA-coated beads (total of 20 ?g of OVA per recipient) into adult B6 mice.
Proliferation of OT-I T cells was assessed 48 h after transfer by CFSE dilution in
CD8?T cells. Data are representative of four to five mice per condition. (F)
Proliferation of transferred, naive BDC2.5 T cells (gated on CD4?V?4?cells) in
PLNs and SLNs after i.p. injection of graded doses of pancreatic islets or PBS into
divided BDC2.5 T cells among the donor CD4?T cells.
Preferential migration to PLNs. (A) Distribution of transferred cells was
www.pnas.org?cgi?doi?10.1073?pnas.0509006102 Turley et al.
To define the specificity of this trafficking route, purified
populations of B and T lymphocytes were used as donor cells to
test whether preferential homing to PLNs is restricted to a
particular lymphocyte subset. Both populations behaved like the
unfractionated splenocytes in their selective migration to PLNs
(Fig. 5A, which is published as supporting information on the
PNAS web site).
We sought to determine whether cellular traffic from the
peritoneum to PLNs was an active process, as might be suggested
by these kinetics. Splenocytes were pretreated with Pertussis
toxin, an inhibitor of G?isignaling that impedes signal process-
ing, and in particular the response to chemotactic cues (20),
before transfer into the peritoneum. Treated donor cells were
unable to selectively access PLNs (Fig. 1D), confirming that the
selective migration to the pancreas is an active process.
To ascertain whether this trafficking pattern was unique to
NOD and?or resulted from ongoing pancreatic inflammation,
identical transfer experiments were carried out with diabetes-
resistant mouse strains. As with NOD, donor cells introduced
into the peritoneum of B6 and B6.H2g7animals also settled
preferentially in the PLNs (Fig. 5B). To determine whether
recipient lymphocytes played any role in establishing this polar-
ized distribution, we transferred wild-type splenocytes into
alymphoid SCID mice. Donor cell distribution in immunodefi-
cient recipients was as skewed as in wild-type recipients, indi-
cating that recipient lymphocytes do not instruct this process
(Fig. 5C). Thus, the selectivity of cellular migration from the
peritoneum to PLNs is a general phenomenon not particular to
any mouse strain or cell type, or to being prone to T1D.
Next, we sought to determine whether preferential trafficking of
cells to PLNs also applies to antigens. We traced the drainage of
several particulate antigens, each administered i.p., through their
ability to elicit the proliferation of antigen-specific T cells. Mice
received chicken OVA- or control-coated microspheres, and pre-
sentation was evaluated by assessing dilution of CFSE label in
OVA-specific T cells from OT-I tg mice (16) transferred i.v. In
animals that received OVA microspheres, OT-I T cells divided
extensively in PLNs after 48 h, to a lesser degree in MLNs, and not
at all in SLNs (Fig. 1E). Infusion of OVA-containing splenocytes
gave similar results, with elevated OT-I activation in PLNs as
compared with other LNs (data not shown). Pancreatic islets were
used as a third form of particulate antigen, probably the form most
directly relevant to diabetes. Here, presentation was measured as
proliferation of naı ¨ve ? cell-reactive CD4?T cells derived from
BDC2.5 TCR tg mice (15) [using the time window between
initiation of traffic from the peritoneum to PLNs, described below,
and the onset of presentation of pancreatic self-antigen at 15 days
12-day-old animals. Within 3 days of islet infusion, transferred T
indicating that antigen-presenting cells transported islet antigens-
derived MHC class II molecule?peptide complexes to PLNs. These
the PLNs of adult mice in a preferential manner.
The presentation of islet-? cell autoantigens in PLNs is a
days of age in mice (2, 3). This observation prompted us to
address the question of when preferential lymphatic access to the
PLNs is established, in particular whether there is any temporal
relationship with the appearance of pancreatic antigens in PLNs.
To that end, labeled splenocytes from adult mice were trans-
ferred into the peritoneal cavities of infant (?1 week old) or
24 h later. The pattern of donor-cell trafficking in the 1-week-old
recipients was strikingly different from that in adults, with no
enhanced traffic to PLNs in the former case (Fig. 2A). This
pattern did not change when the dose of donor cells was varied
(data not shown). To establish more precisely when PLNs
develop into a site of convergence for lymphatic traffic, we
transferred labeled splenocytes into the peritoneum of recipients
ranging in age from 4 to 28 days. The distribution of donor cells
changed markedly between 11 and 18 days of age, showing the
polarization toward PLNs observed in adult recipients (Fig. 2B).
Reciprocal transfers of splenocytes between infant and adult
donors and recipients clearly indicated that trafficking from the
peritoneum to the PLNs is not dictated by the migrating
lymphocytes but by the developmental stage of the host recipient
tissue (Fig. 2C). These data indicate that nonvascular access to
PLNs develops ?12 days after birth. This time frame is strikingly
reminiscent of that when stimulatory dendritic cells presenting
pancreatic autoantigens make their debut.
The peritoneum is the site of developmentally primitive lym-
system populations such as B-1 B cells. Although normally sterile,
the peritoneum may be the first site under attack by enteric
pathogens, commensals, and dietary contents once intestinal bar-
rier function is breached. Given the close connection between the
peritoneum and PLNs demonstrated above, we asked whether
luminal contents of the gastrointestinal tract might also preferen-
tially enter PLNs. To test for this possibility, we fed B6 mice with
cently labeled splenocytes from adult NOD donors were transferred i.p. into
infant and adult NOD recipients and then enumerated 24 h later in PLNs and
SLNs by cytofluorimetry. Data are shown as the number of CFSE?donor cells
(¢) ? 10?3per 1 ? 106recipient LN cells in PLNs and SLNs of both recipients.
Shown are data from seven to eight mice per age group. (B) Donor cell
trafficking to PLNs, MLNs, and SLNs in recipients of different ages was deter-
mined by flow cytometry. The migration index is calculated as the ratio of
ratio of donor cell density in SLNs to the donor cell density in MLNs (open
cytes from adult and infant NOD donors were transferred i.p. into adult and
infant recipients and then enumerated 24 h later in recipient LNs by cytofluo-
rimetry. The migration index is calculated as the ratio of donor cell density in
PLNs to the donor cell density in SLNs.
Lymphatic access to PLNs is developmentally regulated. (A) Fluores-
Turley et al.
December 6, 2005 ?
vol. 102 ?
no. 49 ?
OVA protein and tested whether the antigen had been ferried to
LNs by assessing the response of OVA-specific T cells (again
measuring proliferation as dilution of fluorescent label in OT-I tg
Peyer’s patches, as expected from previous reports (10), but was
doses of fed antigen, the extent of OVA presentation in PLNs was
Similar results were obtained with MHC class II-restricted, OVA-
specific, OT-II tg T cells (17) (Fig. 3B). Therefore, PLNs can be
to present antigens from the gastrointestinal contents.
Given that enteric proteins are presented by antigen-presenting
cells within PLNs, we hypothesized that perturbations occurring in
the gut may also reach the PLNs and affect their ability to present
endocrine self-antigens. For instance, strong T cell responses to gut
antigens in the PLN might enhance bystander responses to pan-
creatic autoantigens; alternatively, enhanced activation of gut an-
of PLN antigen-presenting cells, eliciting more aggressive re-
sponses. To test this hypothesis, we examined the priming of
diabetogenic T cells in NOD mice treated with several agents that
induce diverse forms of intestinal stress or injury. INDO is a
nonsteroidal antiinflammatory drug that reproducibly causes ul-
ceration in the small intestine of mice (and humans) within 24 h of
administration (19). LP diet also perturbs the small intestine by
modifying the composition of the gut flora, and adaptation to the
altered flora elicits histologically detectable changes in the ileal
lining (21). DSS is a sulfated polysaccharide that disturbs perme-
Proliferation of transferred naive BDC2.5 T cells (gated on CD4?V?4?cells) in
PLNs of NOD mice on a LP or control (Ctl) diet or pretreated with 85 mg?kg
INDO, 5% DSS (DSShi), or 2% DSS (DSSlo) as compared with control littermates
(Ctl). T cell proliferation, assessed by CFSE dilution in BDC2.5 T cells, was
reduced 47% by LP (P ? 0.002), 40% by INDO (P ? 0.004), and 54% by 5% DSS
(P ? 0.001) and was enhanced 22% by 2% DSS (P ? 02). Data are shown as the
percentage of divided BDC2.5 T cells among the donor CD4?T cells. Values in
columns depict number of mice per condition. (B) Proliferation of transferred
naive BDC2.5 T cells (gated on CD4?V?4?cells) in popliteal LNs of NOD mice
immunization with BDC2.5 peptide mimic. Data are shown as the percentage
of divided BDC2.5 T cells among the donor CD4?T cells. Values in columns
depict number of mice per condition. (C) Representative histograms of CD44
staining on donor BDC2.5 T cells in PLNs and MLNs of NOD mice after transfer
into mice on 2% DSS or control (CTL) littermates. (D) Frequency of pancreatic
islets with heavy leukocyte infiltration in BDC2.5?NOD mice treated with
percentage of islets with heavy leukocyte infiltration among total islets. Each
symbol represents an individual mouse.
Proliferation of transferred naive OT-I T cells (gated on CD8?V?2?cells) in
60 mg of OVA (IG OVAhi) or 2 mg of OVA (IG OVAlo) or i.v. injection of 2 mg
of OVA (IV OVA) into adult B6 mice as compared with controls. Proliferation
Each symbol represents an individual mouse. (B) Proliferation of transferred
naive OT-II T cells (gated on CD4?V?2?cells) in MLNs and PLNs after intragas-
Each symbol represents an individual mouse.
Gut antigens are presented to CD8?and CD4?T cells in PLNs. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0509006102 Turley et al.
ability of the colonic mucosa, leading to epithelial injury and Download full-text
erosion in a dose- and time-dependent manner (18, 22). CFSE-
labeled T cells from BDC2.5 mice were transferred into NOD mice
that had been pretreated with LP diet, INDO, or DSS (treatments
were stopped at or before transfer to avoid indirect effects on
responder T cells). BDC2.5 T cells proliferated to a lesser extent in
PLNs of mice on LP (47% inhibition on average) or INDO (40%
inhibition) regimens, compared with littermate controls (Fig. 4A).
The effect of DSS varied with the dose. At high doses, it reduced
BDC2.5 T cell activation (54% inhibition); at low doses, DSS
actually accentuated the proliferation (22% increase). This en-
hancement was specific to PLNs: there was no broadening of the
response to pancreatic autoantigen in other locations, ruling out a
systemic mitogenic impact on BDC2.5 T cells by DSS treatment
effect on the response elicited by mimotope peptide in SLNs (Fig.
4B), establishing that the impact of gut-perturbing agents on the
PLN is highly specific. The effect of low doses of DSS also affected
not in the MLN (Fig. 4C). Finally, we asked whether similar
perturbations would also influence insulitis. Consistent with the
results above, DSS enhanced the severity of insulitis in 4-week-old
BDC2.5 mice (Fig. 4D).
Thus, PLNs are at a very peculiar confluence. They sample
self-antigens from the pancreas but also foreign antigens from
the gastrointestinal tract and the peritoneum, and nonspecific
perturbations of gut physiology have a direct and specific impact
on the response of ? cell-reactive T cells. This connection has
strong conceptual implications for our understanding of pan-
creas-directed autoimmunity and its connection to environmen-
tal factors such as gut microbes and food antigens (8, 9, 11). In
particular, the enhancement in diabetogenic T cell priming and
insulitis severity by increased intestinal permeability and mild
enteropathy provides a potential explanation for the special
relationship between T1D and celiac disease. This inflammatory
disorder of the small intestine is provoked by immune reactivity
to gluten-associated proteins, which also have adjuvant activity
(23). CD is very frequent in T1D patients, detectable in 30% of
patients with HLA-DQ2 and 3–10% of diabetic children and
adolescents (14, 24, 25). CD, but also T1D, can be ameliorated
in human patients and in NOD mice by removing gluten-
containing foods from the diet (9, 26). From the present results
it follows that enteropathy, induced by gluten or other dietary
factors, may provide inflammatory stimuli that make their way
to and activate dendritic cells within PLNs. This interaction
might increase an otherwise sluggish response to pancreatic
autoantigens, by promoting the maturation or mobilization of
Preferential homing to PLNs via peritoneal lymphatics was
developmentally regulated and became fully established only after
10 days of age. Intriguingly, this is also the time frame during which
pancreatic antigens are first presented to islet-reactive T cells in
PLNs. Of course, this parallel might simply be a coincidence, but it
could also be that lymphatic connectivity of PLNs may not occur
before this time. We have shown that the initiation of pancreatic
antigen presentation in PLNs is linked to a programmed increase
blockers and accelerated by provoking ? cell death (3). Thus,
antigen availability and lymphatic connectivity might both contrib-
ute to the delayed presentation of pancreatic self in the neonatal
period. Profound alterations take place in the gut around this
transitional period, with marked changes in food intake and bac-
terial flora, and the epithelial barrier (28, 29). One might hypoth-
esize that these events, relayed via immunological or metabolic
cues, drive the establishment of lymphatic connections to PLNs as
well as the increased supply of self-antigen.
In conclusion, the present study defines a fundamental link
between the gastrointestinal tract and PLNs, providing a conduit
for environmental agents to directly modify the immune re-
sponse to pancreatic autoantigens.
M. Petruzzelli for expert instruction in gastric gavage, A. Pinkhasov and
R. Bronson for help with the histology, and members of the Diabetes
by funds from the National Institutes of Health (Grant R01 DK59658)
and by the Joslin Diabetes and Endocrinology Research Center core
facilities (Grant P30 DK36836) and the cores of the Juvenile Diabetes
Research Foundation Centers for Islet Transplantation and on Immu-
nological Tolerance in Type-1 Diabetes at Harvard Medical School.
S.J.T. received fellowship support from the Cancer Research Institute.
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