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Cutting Edge |
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-chain rearrangements this transgenic mouse
spontaneously develops diabetes and that CTLA-4 negatively regulates
diabetes onset. This strongly suggests that onset of diabetes in
BDC2.5/NOD mice is governed by T cell regulation. We addressed the
mechanism of immune regulation in BDC2.5/NOD mice. We find that
activated spleen cells from young, but not old, BDC2.5/NOD mice are
able to transfer diabetes to NOD-scid recipients. We
have used anti-IL-10R to show that the failure of splenocytes from
older mice to transfer diabetes is due to dominant regulation. We
furthermore found that diabetes developed following anti-IL-10R
treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10
plays a key role in the regulation of diabetes onset in this transgenic
mouse. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The nonobese diabetic
(NOD)3 mouse provides
an excellent model of human insulin-dependent diabetes mellitus (IDDM)
(10). In this mouse model, insulitis develops from 4–5 wk
of age and mice develop diabetes from around 12 wk of age. The BDC2.5
TCR-transgenic mouse (11) expresses the rearranged TCR
- and 
-chain genes (V1V4) of a diabetogenic
CD4+ T cell clone, BDC2.5 (12). When
the BDC2.5 NOD TCR-transgenic line is made homozygous for
scid, C-, or
RAG-, the mice spontaneously develop IDDM
at around 3–5 wk of age, whereas on a RAG+
background the incidence is very low (13, 14). This has
led to the suggestion that T cells expressing endogenously rearranged
TCR genes might be playing a role in the prevention of diabetes in
the BDC2.5 TCR-transgenic mouse as has been proposed for experimental
autoimmune encephalomyelitis in myelin basic protein-specific
TCR-transgenic mice (15). We have conducted experiments to
determine whether there is any evidence in the BDC2.5 TCR-transgenic
mouse for active suppression of diabetogenic T cells. It had previously
been shown that although this transgenic mouse does not itself develop
diabetes at high incidence (and then mostly at an advanced age), it was
possible to transfer disease reproducibly into NOD-scid
recipients using Con A-activated spleen cells from the BDC2.5
transgenic mouse (14). This shows that potentially
diabetogenic effector cells must be present in the nondiabetic BDC2.5
transgenic mouse spleen. The possibility that such potentially
diabetogenic T cells are held in check by regulatory cells has been
inferred from studies showing that anti-CTLA-4 Abs can induce IDDM
in very young BDC2.5 TCR-transgenic mice (16). We have
used the Con A activation and transfer system to identify mechanisms
contributing to regulation of these diabetogenic T cells. We found that
disease transfer reproducibly occurred only when donor cells were taken
from young mice and that the ability to transfer disease diminished
with the age of the mouse. Our studies show diabetogenic cells remain
in the spleen cell pool derived from older mice, as treatment of
NOD-scid recipients with Ab to the IL-10R permitted disease
transfer by these cells. A key role for the IL-10R in modulating
IDDM was further shown by the ability of IL-10R-specific Ab to bypass
the requirement for Con A activation of donor splenocytes for
disease transfer and furthermore directly promote the onset of IDDM in
BDC2.5 TCR-transgenic mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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NOD, NOD-scid, BDC2.5/NOD, and BDC2.5/NOD-scid transgenic mice were maintained in the Biological Services facility of the Department of Pathology (University of Cambridge, Cambridge, U.K.). NOD-scid mice or transgenic mice crossed on to the NOD-scid background were maintained in microisolator cages with filtered air.
Abs and reagents
The hybridomas 1B1.3a (anti-IL-10R) and GL113 (isotype control) (rat IgG1) were obtained from Dr. K. Moore (DNAX, Palo Alto, CA). Ab was harvested from culture supernatants by ammonium sulfate precipitation followed by dialysis against PBS. Total protein was estimated from the OD280. Endotoxin levels were <1 EU/mg protein.
Other Abs were obtained from BD PharMingen (Oxford, U.K.) and reagents were obtained from Sigma-Aldrich (Poole, Dorset, U.K.).
In vitro culture of splenocytes
Single cell suspensions of BDC2.5/NOD mouse spleens were
cultured under standard conditions for varying times up to 72 h in
the presence of Con A (5 µg/ml) or islets. For cytokine ELISAs and
FACS staining anti-IL-10R or isotype control Ab preparations were
added (30 µg/ml). For transfer into recipient mice, residual Con A
was removed by addition of methyl--D-mannopyranoside (10
mg/ml), and the cells were washed three times.
CD25+ cell depletion
Splenocytes were depleted of CD25+ cells by incubating with biotinylated anti-CD25 Ab (BD PharMingen) followed by streptavidin Dynabeads (Dynal Biotech, Wirral, U.K.) and passage over a magnet (x2).
Islet isolation
Male NOD mice were sacrificed and the common bile duct was infused in situ with collagenase. The pancreas was then removed and digested at 37°C. The islets were enriched on a Eurocollins-Ficoll gradient and washed in HBSS containing BSA, and islets were handpicked using a siliconized micropipette.
ELISA for IL-10, IL-12 and IFN-
Sera or supernatants were assessed for the presence of cytokines
using a capture ELISA (Opteia anti-mouse cytokine kits; BD
PharMingen). The concentration of cytokine was calculated from graphs
for IL-10, IL-12 and IFN- standard preparations.
Flow cytometric analysis
Cells were stained by standard methods using rat anti-mouse reagents CD4-FITC, CD25-PE, and CD69-PE (BD PharMingen) and analyzed on a BD Biosciences FACScan instrument (BD Biosciences, Oxford, U.K.).
In vivo Ab treatment
In the transfer experiments recipients were treated with 0.5 mg of the Ab preparation (or isotype control) i.p. on days 0, 1, 4, and 7. BDC2.5/NOD mice were given the same dose i.p. on days 0, 5, and 14.
Assessment of diabetes
Recipient mice were tested for the presence of urinary glucose using Diastix (Bayer, Newbury, U.K.). Blood glucose was measured using an Esprit glucometer and glucose test strips (Bayer). Mice were considered diabetic if they had urinary glucose and blood glucose concentrations of 12 mM or above on two occasions.
Histological examination
Pancreases were processed for wax histology. Five-micrometer sections were taken at eight levels (200 µm apart) and stained with H&E. Total islets per section were counted and the degree of cellular infiltration was scored.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Female NOD mice usually develop IDDM from 12 wk of age, achieving
an incidence of around 80% at 30 wk of age. However, diabetes does not
develop in BDC2.5 TCR-transgenic mice at such high incidence
(13). This is not due to an absence of potentially
diabetogenic effector cells, because Con A-activated splenocytes from
6-wk old BDC2.5 TCR-transgenic mice are able to transfer diabetes to
NOD-scid recipients (Ref. 14 and Fig. 1
A). We find that this ability
to transfer IDDM decreases with the age of the donor mouse. Diabetes is
transferred within 14 days by splenocytes from donor mice of less than
10 wk of age, whereas disease transfer is less reliable when activated
splenocytes from older donors are used and the time to disease onset is
extended (Fig. 1A). The impaired ability of splenocytes from
older mice to transfer disease is not due to their failure to traffic
to the islets, as splenocytes from older BDC2.5/NOD mice caused a
significant degree of insulitis in NOD-scid recipients (Fig. 1, B and C).
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As it was possible that there was an increased ratio of regulatory
to effector cells in the spleens of older mice, we looked for evidence
for regulatory cell-mediated inhibition of disease transfer. It has
been shown that CD4+CD25+ T
cells are able to down-regulate autoreactive or inflammatory responses
(17, 18, 19, 20, 21).
CD4+CD25+ T cells are
present only in very low numbers (0.9%) in spleens of BDC2.5
NOD-scid mice, whereas they are clearly present in the
spleens of NOD and BDC2.5 TCR-transgenic mice (10.2% and 5.6%
respectively). All BDC2.5 NOD-scid mice develop disease when
3–5 wk old, whereas BDC2.5 TCR NOD transgenic mice do not develop
diabetes at a high incidence. To determine whether the presence of
CD4+CD25+ T cells
contributed to this low incidence of diabetes we depleted
CD25+ cells from the splenocyte pools of older
transgenic mice before Con A activation and examined their ability to
transfer disease. Depletion of CD25+ cells failed
to enable diabetes transfer by activated splenocytes from older mice
(zero of four mice in each of the depleted and control groups).
Comparable levels of insulitis developed in NOD-scid
recipients of both depleted and nondepleted spleen cells (Fig. 2A). Therefore, there is no
evidence for CD25+ T cell-mediated prevention of
diabetes transfer in this experimental system. This result parallels
the observation in the rat where peripheral CD4+
T cells, which prevent diabetes, are found in both
CD25+ and CD25-
populations (22).
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Because IL-10 has been implicated in regulation of inflammation
and autoimmunity (23), we examined whether Ab which is
specific for, and blocks, the IL-10R (24) influenced the
ability of activated splenocytes from older mice to transfer diabetes.
Fig. 2
B shows that these are able to cause diabetes in
anti-IL-10R-treated NOD-scid recipients. These data
suggest that interactions through the IL-10R play a key role in the
regulation of type 1 diabetes in BDC2.5/NOD mice.
It was not possible to transfer IDDM with nonactivated splenocytes from
young BDC2.5 TCR-transgenic mice (14). To determine
whether this failure to transfer disease also involved interactions
through the IL-10R we transferred splenocytes from young BDC 2.5
TCR-transgenic mice without Con A activation into NOD-scid
recipients in the presence or absence of anti-IL-10R. Treatment of
recipients with anti-IL-10R led to the development of IDDM (seven
of nine compared with zero of nine control mice). This was also seen
when spleen cells from older BDC2.5 TCR-transgenic mice were used
(eight of eight compared with zero of eight control mice). To determine
whether the anti-IL-10R had caused direct T cell activation, spleen
cells were cultured with IL-10R Ab and analyzed by FACS for expression
of CD25 and CD69. There was no evidence that anti-IL-10R caused T
cell activation (Fig. 2C). A further indication that
anti-IL-10R did not directly activate diabetogenic effector cells
is provided by our observation that BDC 2.5 NOD-scid mice
treated with control or anti-IL-10R Ab develop type 1 diabetes with
comparable kinetics (data not shown).
The ability of IL-10R Ab to promote disease in these transfer
experiments suggested that such cytokine-mediated control might account
for the low incidence of diabetes in BDC2.5 TCR-transgenic mice.
BDC2.5/NOD mice (6 wk) were given three injections of either
anti-IL-10R or control Ab and monitored for disease onset. The
anti-IL-10R Ab-treated mice started to develop diabetes at 11 wk of
age, and by 23 wk 100% were diabetic. None of the control mice
developed diabetes and remained disease free for >30 wk (Fig. 3A). These data strongly
suggest that in vivo an IL-10-like cytokine plays a regulatory role in
preventing diabetes development in BDC 2.5 TCR-transgenic
mice.
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As early as 24 h following one injection of anti-IL-10R,
serum levels of IL-12 were elevated. This increased IL-12 was sustained
over the assessment period of 13 days (Fig. 3B). The
presence of elevated serum IL-12 suggested that IL-10R Ab treatment was
favoring a proinflammatory response. Therefore, we assessed whether
anti-IL-10R could influence the production of proinflammatory
cytokines in vitro. BDC2.5/NOD splenocytes were cultured for 48 h
with Con A in the presence of anti-IL-10R or control Ab. The
production of both IL-12 and IFN- was increased when the
TCR-transgenic splenocytes were cultured in the presence of Con A and
anti-IL-10R in vitro (Fig. 3C). Comparable experiments
were conducted using isolated NOD islets to stimulate T cell responses.
As shown in Fig. 3D, there was an increased
production of IL-12 and IFN- when BDC2.5/NOD spleen cells were
cultured in the presence of an anti-IL-10R compared with an isotype
control Ab. These data strongly suggest that anti-IL-10R
facilitates the onset of diabetes by permitting the expression of a Th1
response against islets and that the development of diabetes in
BDC2.5/NOD mice is endogenously regulated by interactions through this
receptor.
A regulatory population of T cells which both produces and responds to
IL-10 has been identified in both humans and mice (8, 25).
T cells are clearly involved in the regulatory process that prevents
diabetes in BDC2.5/NOD mice. It is possible that these cells use a TCR
with an endogenously rearranged -chain, because they are not present
in BDC2.5/NOD-scid mice. By producing IL-10 such regulatory
T cells could influence the development of type 1 diabetes in many
ways. Th1 cells, dendritic cells (DC), and macrophages play a role in
the development of diabetes (26). IL-10 is known to be
able to inhibit both DC and macrophage function. IL-10 has been shown
to be an autocrine growth factor for DC, and neutralization of IL-10,
together with a DC maturation signal, has been shown to increase
expression of CD80, CD86, and MHC, as well as production of IL-12 and
TNF-. Anti-IL-10R treatment of BDC2.5/NOD mice resulted in elevated
serum IL-12 levels (Fig. 3B) while its inclusion in vitro
clearly enhanced both IL-12 and IFN- production in response not only
to Con A but also to islets (Fig. 3, C and D).
Prevention of an IL-10/IL-10R interaction in BDC2.5/NOD mice would
therefore be predicted to skew the T cell response to Th1 and thus be
potentially diabetogenic. IL-10 inhibits production of a range of
inflammatory mediators, including IL-1, IL-1, and TNF-, by
macrophages (27, 28). These cytokines, together with
IFN-, have been shown to be particularly damaging to pancreatic
cells (29). Inhibition of macrophage function would
therefore be expected to provide additional protection from autoimmune
destruction.
There are several studies showing that administration of exogenous
IL-10 (as IL-10 itself, an IL-10/Fc fusion protein, or a viral homolog
of IL-10) is able to prevent development of insulitis or diabetes onset
in NOD mice (30, 31, 32). Furthermore, transfer of
cell-specific T cell clones transduced with IL-10 into NOD mice
prevented type 1 diabetes (33). However, it would seem
that the timing of exposure to IL-10 critically influences the outcome
of exposure to this cytokine, because expression of IL-10 in the
pancreas from an early stage through transgenesis exacerbated disease,
possibly by influencing extravasation of lymphocytes into the pancreas
(34). Anti-IL-10R induces diabetes in BDC2.5/NOD mice if
given when the transgenic mice are 6 wk of age. This contrasts with the
effects of anti-CTLA-4 on BDC2.5/NOD mice where Ab treatment was
only effective if given to mice <19 days of age and before the
development of insulitis. Because BDC2.5/NOD mice deficient in CTLA-4
spontaneously develop IDDM at high incidence from 5 wk of age
(35), this suggests that T cells expressing CTLA-4 are
required to play their regulatory role at an early stage in the
evolution of diabetes in NOD mice. Our studies have shown that while
anti-IL-10R has a comparable effect to that of anti-CTLA-4, it
is able to act at later time points, further highlighting a key role
for IL-10 in the regulation of type 1 diabetes in these mice. Our
findings indicate that the anti-IL-10R Ab permits the emergence of
a Th1 response to islet Ags and that under normal circumstances
regulatory T cells use interactions through this receptor to
inhibit the development of diabetes in BDC2.5/NOD mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anne Cooke, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. E-mail address: ac{at}mole.bio.cam.ac.uk
3 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; DC, dendritic cell.
Received for publication August 21, 2001. Accepted for publication October 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlowCD4+ T cells. J. Exp. Med. 183:2669.-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
and IL-10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166:5530. cells in nonobese diabetic mice. J. Exp. Med. 179:1379.This article has been cited by other articles:
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  D. C. Thomas, R. J. Mellanby, and A. Cooke Comment on: Tritt et al. (2007) Functional Waning of Naturally Occurring CD4+ Regulatory T-cells Contributes to the Onset of Autoimmune Diabetes: Diabetes 57:113-123, 2007 Diabetes, March 1, 2008; 57(3): e6 - e6. [Full Text] [PDF]
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 F. J. Ward, A. M. Hall, L. S. Cairns, A. S. Leggat, S. J. Urbaniak, M. A. Vickers, and R. N. Barker Clonal regulatory T cells specific for a red blood cell autoantigen in human autoimmune hemolytic anemia Blood, January 15, 2008; 111(2): 680 - 687. [Abstract] [Full Text] [PDF]
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 K. A. Smith, S. Efstathiou, and A. Cooke Murine Gammaherpesvirus-68 Infection Alters Self-Antigen Presentation and Type 1 Diabetes Onset in NOD Mice J. Immunol., December 1, 2007; 179(11): 7325 - 7333. [Abstract] [Full Text] [PDF]
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 K. A. Saunders, T. Raine, A. Cooke, and C. E. Lawrence Inhibition of Autoimmune Type 1 Diabetes by Gastrointestinal Helminth Infection Infect. Immun., January 1, 2007; 75(1): 397 - 407. [Abstract] [Full Text] [PDF]
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D. Bruder, A. M. Westendorf, W. Hansen, S. Prettin, A. D. Gruber, Y. Qian, H. von Boehmer, K. Mahnke, and J. Buer On the Edge of Autoimmunity: T-Cell Stimulation by Steady-State Dendritic Cells Prevents Autoimmune Diabetes Diabetes, December 1, 2005; 54(12): 3395 - 3401. [Abstract] [Full Text] [PDF]
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R. K. Gregg, J. J. Bell, H.-H. Lee, R. Jain, S. J. Schoenleber, R. Divekar, and H. Zaghouani IL-10 Diminishes CTLA-4 Expression on Islet-Resident T Cells and Sustains Their Activation Rather Than Tolerance J. Immunol., January 15, 2005; 174(2): 662 - 670. [Abstract] [Full Text] [PDF]
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N. Sarween, A. Chodos, C. Raykundalia, M. Khan, A. K. Abbas, and L. S. K. Walker CD4+CD25+ Cells Controlling a Pathogenic CD4 Response Inhibit Cytokine Differentiation, CXCR-3 Expression, and Tissue Invasion J. Immunol., September 1, 2004; 173(5): 2942 - 2951. [Abstract] [Full Text] [PDF]
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 A. E. Herman, G. J. Freeman, D. Mathis, and C. Benoist CD4+CD25+ T Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic Lesion J. Exp. Med., June 7, 2004; 199(11): 1479 - 1489. [Abstract] [Full Text] [PDF]
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 A. R. Mathers and C. F. Cuff Role of Interleukin-4 (IL-4) and IL-10 in Serum Immunoglobulin G Antibody Responses following Mucosal or Systemic Reovirus Infection J. Virol., April 1, 2004; 78(7): 3352 - 3360. [Abstract] [Full Text] [PDF]
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S. Turley, L. Poirot, M. Hattori, C. Benoist, and D. Mathis Physiological {beta} Cell Death Triggers Priming of Self-reactive T Cells by Dendritic Cells in a Type-1 Diabetes Model J. Exp. Med., November 17, 2003; 198(10): 1527 - 1537. [Abstract] [Full Text] [PDF]
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Y. C. Zhang, A. Pileggi, A. Agarwal, R. D. Molano, M. Powers, T. Brusko, C. Wasserfall, K. Goudy, E. Zahr, R. Poggioli, et al. Adeno-Associated Virus-Mediated IL-10 Gene Therapy Inhibits Diabetes Recurrence in Syngeneic Islet Cell Transplantation of NOD Mice Diabetes, March 1, 2003; 52(3): 708 - 716. [Abstract] [Full Text] [PDF]
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A. M. Hall, F. J. Ward, M. A. Vickers, L.-M. Stott, S. J. Urbaniak, and R. N. Barker Interleukin-10-mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen Blood, December 15, 2002; 100(13): 4529 - 4536. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Six weeks; 
, 8 wk; 
, 11–15 wk; x, 20 wk.
B, Adult NOD-scid recipients of Con
A-activated splenocytes from 20-wk old donors which did not develop
diabetes were killed at the end of the experiment (i.e., day 40), and
their pancreases were taken for histological analysis. This histogram
shows the mean percentage of islets with a given degree of cellular
infiltration. No bar, No infiltration; 
, peri-islet infiltration;
light gray bar, up to 10% infiltration; 
, 10–25% infiltration;
, 25–50% infiltration; 
