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Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8603, Université Paris V, Hôpital Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and no IL-4 and IL-10 upon in vitro stimulation.
Consistently, the inhibition of diabetes transfer was not mediated by
IL-4 and IL-10. Regulatory cells homed to the pancreas and modified the
migration of diabetogenic to the islets, which resulted in a decreased
insulitis severity. The efficiency of CD62L+ T cells was
dose dependent, independent of sex and disease prevalence. Protection
mechanisms did not involve the CD62L molecule, an observation that may
relate to the fact that CD4+ CD62Lhigh lymph
node cells were less potent than their splenic counterparts. Regulatory
T cells were detectable after weaning and persist until disease onset,
sustaining the notion that diabetes is a late and abrupt event. Thus,
the CD62L molecule appears as a unique marker that can discriminate
diabetogenic (previously shown to be CD62L-) from
regulatory T cells. The phenotypic and functional characteristics of
protective CD4+ CD62L+ cells suggest they are
different from Th2-, Tr1-, and NK T-type cells, reported to be
implicated in the control of diabetes in NOD mice, and may represent a
new immunoregulatory population. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effector T cells, characterized by their ability to transfer diabetes in immunodeficient recipients, are found in different organs of diabetic mice (3) and in prediabetic mouse infiltrated islets (4) and express the phenotype of activated/memory T cells: low levels of CD62L (5) and CD45RB (6).
Several lines of evidence show the presence of regulatory T cells in
the NOD mouse. Cyclophosphamide induces acute insulin-dependent
diabetes mellitus in young NOD mice (7, 8); sublethal
irradiation (9) or thymectomy and CD4 cell depletion
(10) are required to transfer disease by diabetogenic T
cells. In NOD females, thymectomy at weaning prevents the generation of
protective cells and thus accelerates the onset of diabetes
(11). CD4 T cells from nondiabetic young mice prevent the
transfer of diabetes by splenocytes from diabetic mice (6, 12, 13). Regulatory cells are also present in the thymus of young
healthy mice and, thus far, have been best characterized. They belong
to at least two subpopulations. Mature CD4+
CD62L+ thymocytes (14) and TCR
ß+ CD4-
CD8- double negative thymocytes
(15) were found to be capable of inhibiting the transfer
of diabetes. The latter thymocyte population includes NK T cells, and a
role for NK T spleen cells in the regulation of diabetes development
(16) has recently been suggested. These findings are
compatible with the existence of more than one type of regulatory T
cells, as already observed in aerosol-induced tolerance (17, 18) and in the CD45RBlow T cell subset of
normal mice (19, 20). CD4+ and
CD8+ T cell clones derived from islet infiltrates
were found to prevent the transfer of diabetes, suggesting that the
impact on final disease expression of such T cells may result from
local regulation. Lastly, in transgenic NOD mice in which
CD4+ cells express a diabetogenic TCR, BDC2.5
disease prevalence is lower than in BDC2.5/NOD-scid mice, indicating
the presence of protective cells with endogenous TCR in BDC2.5/NOD and
their absence in BDC2.5/NOD-scid mice (21).
A number of attempts to keep self-reactive T cells in check have succeeded in preventing diabetes in NOD mice. In particular, regulatory T cells that suppress the disease can be induced by injection of candidate autoantigens (insulin, glutamic acid decarboxylase 65, heat shock protein 60) (22, 23, 24, 25). These cells may exert their function through the cytokines they secrete (i.e., IL-4, IL-10, TGF-ß) and/or other inhibitory activities. Indeed, treatment of NOD mice with, or transgenic expression in the pancreas of, certain cytokines abrogate diabetes development (reviewed in Ref. 26).
In unmanipulated NOD mice, the phenotypic and functional characteristics of regulatory CD4+ T cells present in the spleen during the prediabetic period await further exploration. We report here an analysis of such cells and discuss their mechanism(s) of action.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD-Nk (Nk for Necker), NOD-NON-Thy1a and NOD-scid mice were bred in our own facilities under specific pathogen-free conditions. Recipients were NOD-scid mice aged 4–6 wk except in two experiments where male NOD-Nk mice irradiated with 7.5 Gy on the day before cell transfer were used. Diabetic NOD-Nk or NOD.NON-Thy-1a mice were pooled and used within the week following disease onset. Similar numbers of NOD-Nk and of NOD.NON-Thy-1a diabetogenic cells transfer diabetes with the same kinetics.
IL-4-deficient NOD mice were obtained from D. Serreze, (The Jackson
Laboratory, Bar Harbor, ME), and TCR -chain-deficient NOD mice (NOD
C-/-) were provided by C. Benoist and D.
Mathis (Centre Universitaire, Strasbourg, France).
Donor mice for protective cells were females or males aged 4–10 wk, with a majority of experiments performed with 5- to 6-wk-old animals.
Abs and flow cytometry
Monoclonal Abs to CD62L (MEL-14), TCR ß (H57-597), CD4 (GK
1.5), CD8 (53-6.7), Thy-1.2 (30-H.12), B220 (RA3-6B2), and GR1
(RB6–8C5) were obtained from ascites. Abs were used either purified or
conjugated to FITC or biotin. Anti-CD4 (CT-CD4), anti-CD8 (CT-CD8),
anti-B220 (RA3-6B2), and anti-Mac-1 (M1/70.15) were purchased
as PE conjugates from Caltag (Tébu, France). PE-conjugated
anti-CD45 (30F11.1) and streptavidin were obtained from PharMingen
(Grenoble, France). Anti-Thy-1.1 (MRC OX-7) (Cedarlane, Tébu,
France) was used for immunohistochemistry. Biotin- and HRP-conjugated
mouse anti-rat IgG and FITC-anti-rat 
-chain (MARK-1)
(Immunotech, Marseille, France) were used as second-stage Abs.
Extravidin-FITC was obtained from Sigma (L’Isle d’Albeau Chesnes,
France).
Cells from spleens, pooled mesenteric and peripheral lymph nodes (LN), bone marrow, and islet infiltrates (27) were analyzed using a FACScan or a FACScalibur (Becton Dickinson, Grenoble, France).
Cell fractionation
Immunomagnetic cell sorting was used for all cell purifications. To purify CD62L+ and CD62L- cell populations, cell suspensions were incubated with biotinylated MEL-14 Ab for 20 min on ice, washed, and further incubated with streptavidin microbeads (Miltenyi Biotec, Tébu, France). Cells were separated using a VarioMACS device according to the manufacturer’s protocol. The retained (CD62L+) and nonretained cells (CD62L-) were stained with a mixture of FITC-anti-CD4, FITC-anti-CD8, and PE-streptavidin to assess their purity. FACS analysis showed that CD62L+ cells reproducibly comprised about 50% T cells of which 2–3% expressed low levels of CD62L. The negative fraction contained 30–40% T cells that were 99% CD62L-.
CD4 and CD8 spleen cells were purified using anti-CD4- and anti-CD8-coated microbeads (Miltenyi Biotec), respectively. Purified B cells were obtained by immunomagnetic cell sorting of spleen cells successively labeled with biotinylated anti-B220 and streptavidin microbeads. Likewise, granulocytes were positively selected from bone marrow cells using biotinylated anti-GR1. To enrich for CD4+ CD62L+ cells, spleen cell suspensions were first depleted in B cells, macrophages, and CD8+ cells. Subsequently, CD62L+ cells were positively selected. The final cell population contained 80% T cells, all of which being CD4+, CD62L+ cells.
Adoptive transfer of diabetes
Diabetogenic T cells were prepared from the spleens of at least
five recently diabetic NOD-Nk or
NOD-NON-Thy1a mice. We have previously shown that
diabetogenic T cells were exclusively comprised within the
CD62L- T cell population (5). Thus,
the cell suspension was stained for CD4, CD8, and CD62L to define the
percentage of CD62L- T cells. To normalize
diabetes transfers, the number of spleen cells injected was calculated
so that each recipient received i.v. 4 x
105 CD62L- T cells
(corresponding to 
1.2 x 106 T cells or
3 x 106 spleen cells).
To test the regulatory function of cell subpopulations, cotransfer experiments were performed. NOD-scid recipients were injected with 4 x 105 CD62L- T cell equivalents from diabetic mice (referred to as diabetogenic T cells) and various numbers of test cells.
Recipients (4–12 mice/group) were tested weekly for glycosuria until 12 wk after transfer. After a positive urine test, blood glucose levels were determined (Reflolux, Boehringer Mannheim, Mannheim, Germany). Animals showing glycemia >300 mg/dl at two consecutive measurements were classified as overtly diabetic.
In vivo Ab treatment of recipient mice
To determine the role of CD62L in the protection from diabetes transfer, recipients were injected with donor spleen cells incubated for 30 min on ice with purified anti-CD62L before the i.v. injection of the cell and Ab mixture (300 µg/mouse) into NOD-scid mice. Mice were further injected i.p with 300 µg/mouse MEL-14 three times a week. Control mice received PBS in one experiment and purified rat IgG (Sigma) in a second one.
Recipients that were treated with neutralizing anti-cytokine Abs were injected on the day of transfer (day 0), and days 2 and 4 after transfer with either 11B11 or JES5–2A5 or a mixture of both neutralizing Abs (500 µg/mouse/Ab) and with 300 µg of each Ab twice a week for 8 wk. Control mice received purified rat IgG. Using similar protocols, 11B11 and JES5–2A5 Abs were found effective in autoimmune diabetes (15, 28).
Staining of cryostat sections
Cryostat sections of pancreas (5 µm) were fixed in cold acetone for 10 min and air-dried for 20 min. Sections were rehydrated in PBS containing 5% FCS for 20 min and further incubated with primary purified anti-CD4 or a mixture of purified anti-Thy-1.2 and biotinylated anti-Thy-1.1 in PBS for 30 min. After three washes, sections labeled with primary anti-CD4 were revealed by HRP-conjugated mouse anti-rat IgG diluted in PBS plus 5% mouse serum. The sections labeled with the mixture of Abs were incubated with a mixture of HRP-mouse anti-rat IgG and avidin-biotinylated alkaline phosphatase complex (Vectastain ABC-AP kit; Vector, Biosys, Compiégne, France). Peroxidase activity was first detected using 3-amino-9-ethyl-carbazole (Sigma) as a substrate, while the alkaline phosphatase substrate was 5-bromo-4-chloro-indolylphosphate/nitroblue tetrazolium. Sections with immunoperoxidase staining were counterstained with hematoxylin.
Cytokine ELISA
The equivalent of 105 T cells from
unfractionated and purified populations were stimulated on plate-bound
anti-TCR-ß (0.5 µg/well) in 96-well culture plates for
48 h at 37°C. The supernatants were assayed for IFN-, IL-4,
and IL-10 content using AN18 (anti-IFN-), 11B11 (anti-IL-4),
and JES5-2A5 (anti-IL-10) as the capture Abs, and
biotinylated-R4-6A2 (anti-IFN-), -BVD6 (anti-IL-4), and
-SXC-1 (anti-IL-10) (PharMingen) as the detection Abs.
The presence of IL-2 was determined by the induction of CTLL proliferation with test supernatants or recombinant IL-2 for a total of 48 h. The culture were pulsed with [3H]TdR for the last 18 h. The TGF-ß content of the supernatants was determined using an ELISA kit (Genzyme, Cambridge, MA).
Statistical analysis
Pooled data were computed as mean ± SEM and compared using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T cells from diabetic mice able to rapidly transfer diabetes into immunoincompetent recipients are present in the CD62L- cell population, while CD62L+ T cells are poorly efficient in inducing the disease (5). This observation led us to test whether the CD62L+ T cell population contains the immunoregulatory activity that keeps pathogenic T cells from attacking pancreatic islets during the prediabetic period. We compared the efficiency of unfractionated and CD62L+ T cell-depleted splenocytes from 4- to 10-wk-old prediabetic mice to induce diabetes. At all ages tested, T cells depleted in CD62L+ cells transferred diabetes earlier than unseparated T cells and/or in a higher percentage of recipients, suggesting that CD62L+ cells delayed the diabetogenic autoimmune process (data not shown).
This hypothesis was confirmed in cotransfer experiments where
pathogenic T cells were injected together with purified
CD62L+ spleen cells from prediabetic mice into
NOD-scid hosts. The protective effect of total spleen T cells (20
x 106) was compared with that of
CD62L+ (15 x 106) and
CD62L- (4 x 106) T
cells (the numbers of purified cells were deduced from their
physiological proportions) from 6-wk-old female mice. While
CD62L- T cells did not protect from transfer of
diabetes, CD62L+ T cells delayed the onset of the
disease by 6 wk compared with mice receiving diabetogenic T cells only
and were more efficient than total T cells (Fig. 1
). The absence of protective effect of
CD62L- T cells was confirmed in a second
experiment where 107
CD62L- T cells were tested (data not shown).
CD62L+ T cells also protected from transfer of
diabetes in two independent experiments using irradiated male NOD
recipients, and the delay lasted longer (8 and 11 wk, respectively)
than in NOD-scid hosts (data not shown). This prolonged protection may
result from the additional effect of regulatory T cells newly produced
by the thymus.
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) and were used in subsequent
experiments.
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We examined whether, in agreement with reports from several groups
(6, 12, 13), the CD62L+ regulatory
spleen T cells were CD4+ cells. While purified
CD8 spleen cells had no effect, 15 x 106
purified CD4+ cells or total spleen cells
containing 15 x 106
CD4+ cells prevented the transfer of diabetes by
diabetogenic T cells. In addition, coinjection of 10 x
106 CD4+
CD62L+ cells from prediabetic animals with
diabetogenic T cells inhibited the transfer of diabetes more
efficiently than total CD4 cells (Fig. 3). Cotransfer of spleen B cells or bone
marrow granulocytes from young NOD mice or splenocytes from NOD
C-/- mice had no effect on diabetes transfer
(Table I), showing that protection was
not due to a dilution effect of the diabetogenic population and the
specific role of CD4+
CD62L+ cells in the control of pathogenic cells
in young prediabetic mice.
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The protective capacity of CD62L+ spleen T cells from prediabetic NOD mice was demonstrated in mice as young as 4 wk old and observed in 14 of 14 experiments using mice from 4 to 10 wk of age. In contrast, when CD62L+ spleen T cells (15–20 x 106/mouse) from recently diabetic animals were coinjected with 4 x 105 diabetogenic T cells, diabetes onset was delayed for several weeks in two of five cotransfer experiments (data not shown). These observations suggest that regulatory T cells are present soon after weaning, and the breakdown of immunoregulation takes place shortly before diabetes onset.
Regulatory T cells localized in islets differently from diabetogenic T cells and modified the homing of the latter
Cotransfer experiments using diabetogenic cells from mice
expressing Thy-1.1 and protective cells from prediabetic mice
expressing Thy-1.2 were performed to visualize the location of both
cell types in the islets. Besides T cells, Thy-1 Ag is expressed at
varying levels by endothelia cells, neurons, muscle cells, and
different connective tissue elements (29, 30). We observed
that anti-Thy-1.2 labeled some vessels in the exocrine tissue and
elements of the connective tissue notably at the periphery of the
inflamed islets in pancreas sections of NOD-scid recipients. This
background (red-brown staining) is exemplified in Fig. 4A, which shows an islet
section from a mouse injected with diabetogenic cells (blue staining)
alone. Labeling with anti-CD4 of semiserial sections decorated both
Thy-1.1+ and Thy-1.2+
CD4+ cells and helped to localize this
unavoidable background and to estimate the relative proportions of
diabetogenic (blue cells in Fig. 4C) and protective T
cells.
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A). When
CD62L+ protective cells were injected alone, they
preferentially located at the periphery of the islets and remained
there for at least 12 wk (Fig. 4, E and F). Thus,
diabetogenic and regulatory T cells differ by their islet homing
capacity. In the pancreas of mice cotransferred with diabetogenic and
protective cells from mice congenic at the Thy-1 locus, both cell types
were present with a high predominance for the protector cells over rare
diabetogenic T cells, and the majority of both types of infiltrating
cells were at the periphery of the islets (Fig. 4C).
CD4+ cells represented a high proportion of
infiltrating cells (Fig. 4, B and D–F). Thus,
protective cells altered the migration of pathogenic T cells to the
islets: both the location and the number of diabetogenic cells were
modified. Homing of regulatory T cells in islets does not involve the CD62L molecule
In two independent experiments, islet infiltrating cells were
isolated from recipients cotransferred 6 wk earlier. The diabetogenic
and protective-type T cells, expressing different Thy-1 alleles,
comprised only a mean of 3 and 6% CD62L+ cells,
respectively (vs 99% for the injected protective cells), and their
ratio was 1:10, similar to that of the injected cells. The lack of
CD62L on regulatory T cells within the islets raised the question of
whether these cells use CD62L to enter into the pancreas. To address
this issue, recipient mice were injected with effector and protective
cells coated with MEL-14 Ab (anti-CD62L) and treated once a week
with 300 µg of MEL-14 Ab until the end of a first experiment. In a
second experiment, mice were treated three times a week. This treatment
induced a clear and long-lasting down-regulation of CD62L expression on
T cells. Thirteen weeks after cell transfer, 80–85% spleen T cells in
MEL-14-treated animals expressed low levels of or no CD62L, vs 30% in
control hosts. In both experiments, mice coinjected with effector and
protective cells and treated with MEL-14 Ab remained free of diabetes
for as long as control mice injected with rat IgG (Fig. 5). These data indicate that homing of
regulatory T cells into the pancreas is CD62L independent.
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). Because regulatory LN T cells
appear less numerous and/or less efficient than
CD62L+ spleen T cells, migration of regulatory T
cells seems to be preferentially directed toward the spleen, making it
a better source of regulatory cells than LN. In addition,
immunohistochemical analysis of recipients’ pancreas showed that
CD62L+ LN T cells localized at the periphery of
the islets as CD62L+ spleen T cells did (data not
shown). Thus, the difference in the ability of T cells from LN and
spleen to protect against diabetes transfer is not related to their
ability to home to the pancreas.
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The cytokine profiles of T cell populations separated on the basis
of CD62L expression were determined after stimulation on plate-bound
anti-TCR-ß Ab. In prediabetic donor mice,
CD62L+ T cells (the protective cells) produced
the type 1 cytokines IFN- and IL-2 (13 ± 7 ng and 3
IU/105 T cells, respectively), but the levels of
the type 2 cytokines IL-4 and IL-10 were generally below the lower
limit of detection (0.1- 0.2 ng/ml). In contrast,
CD62L- T cells secreted high amounts of IFN-,
IL-4, IL-10, and IL-2 (187 ± 40, 4.6 ± 1.2, and 13 ±
2 ng and 33 ± 9 IU/105 T cells,
respectively), which were statistically different from those produced
by CD62L+ T cells (p <
0.02 for all cytokines) (Fig. 7). Similar
results were obtained with spleen cell populations from diabetic mice
(data not shown). The levels of TGF-ß were in the order of 0.5–1
ng/105 cells in all supernatants tested.
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A). In
addition, CD62L+ T cells from IL-4-deficient NOD
mice delayed diabetes transfer as efficiently as
CD62L+ T cells from wild-type mice, confirming
that IL-4 is not involved in protection (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and no IL-4 or IL-10, as opposed to
CD62L- T cells that produced a wide range of
cytokines. Consequently, regulatory cells exerted their function
independently of IL-4 and IL-10, as shown by the absence of effect of
anti-cytokine Ab treatment. In contrast, treatment of recipients
with anti-CD62L Ab showed that the mechanisms involved in both
diabetes transfer (5) and its protection were independent
of CD62L.
We have previously shown that the pathogenic T cells able to rapidly
transfer diabetes are found in the CD62L-/low T
cell population (5). Our demonstration that
CD4+ CD62L+ T cells from
prediabetic NOD mice comprise regulatory T cells able to protect
against diabetes transfer by pathogenic T cells in NOD-scid mice points
to the CD62L molecule as the first marker that can discriminate
Ag-experienced diabetogenic cells from regulatory CD4 T cells. These
regulatory T cells are found among a pool of cells that harbor
phenotypic (CD62L+) and functional (low
production of IL-2 and IFN- and no IL-4 or IL-10 upon in vitro TCR
stimulation) characteristics of naive cells. CD62L expression is
consistent with a naive phenotype (CD62high),
while Ag-exposed cells are CD62-/low
(31); however, long-lived memory lymphocytes were shown to
be CD62L+ (32, 33).
The dormant or activated state of regulatory T cells in diabetes-prone individuals is an unresolved issue. Protective T cells in prediabetic NOD mice are contained in both CD45RBhigh and CD45RBlow CD4+ T cell subsets (6). While the vast majority of CD45RBhigh T cells express CD62L and are naive cells, a small fraction of previously activated CD45RBlow T cells are CD62L+. In type 1 diabetic patients regulatory T cells able to down-modulate the autoimmune response to ß cell membrane Ag-expressed CD45RO and CD45RA, which define memory and naive cells, respectively (34). In normal animals, organ-specific autoimmune diseases can be induced by thymectomy of mice at 3 days of age (35), adult thymectomy and low-dose irradiation of normal rats (36), or transfer of naive CD4+ CD45RBhigh cells to immunodeficient mice (37). These manifestations can be prevented by transplantation of T cell subsets that express a phenotype associated to Ag-experienced T cells: CD4+ CD25+ (35) or CD4+ CD45RBlow in mice (37) and CD4+ CD45RClow in rats (20). These data were unanimously interpreted as evidence that Ag-primed regulatory T cells can prevent naive autoreactive T cells from being activated in normal animals. However, all the regulatory CD4+ CD25+ cells are CD45RBlow (35) and 50% express CD62L (38). Thus, to better define the activation state of regulatory T cells in NOD mice, the coexpression of CD62L with other activation markers such as CD45RB, CD44, and CD25 must be studied.
In the NOD mouse, regulatory spleen T cells express high levels of CD62L, as CD4+ CD62L+ protector thymocytes do (14). The thymocytes that protect PVG RT1 rats from diabetes transfer express the same phenotype (39). Mature CD4+ CD62L+ thymocytes, in both models, are more potent than their spleen cell counterparts. Indeed, protection from diabetes transfer requires about 5–10 times less mature thymocytes than splenocytes. The suggestion by Modigliani et al. (40) that regulatory T cells leave the thymus early and during a limited time implies that after this wave protective cells are increasingly diluted with newly exported naive T cells. In addition, the possibility that regulatory T cells constitute a particular subset of Ag-experienced cells that express CD62L may be envisaged. The paucity of LN in regulatory T cells may result from their preferential homing to the spleen as compared with LN. To exert their regulatory function, protective T cells need to encounter effector T cells. Because entry into LN is dependent upon CD62L (41), effector cells that do not express CD62L will preferentially migrate to sites where lymphocyte homing is CD62L independent (5, 42) (e.g., the spleen, the islets). Thus, regulatory T cells do not need to home to LN, and consequently the treatment with MEL-14 Ab, which prevents lymphocyte homing to LN but not to the spleen, had no effect on the protection. In the islets of mice injected with diabetogenic and protective CD62L+ T cells, infiltrates were located at the periphery of the islets and protective type CD4+ T cells largely exceeded diabetogenic T cells. In contrast, in recipients only injected with diabetogenic T cells, a high number of the latter cells invaded the islets. Hence, regulatory T cells did not inhibit insulitis development, but decreased its severity by restricting the number and preventing the entry of pathogenic T cells into the islets.
Cotransfer of CD62L+ cells delayed diabetes development for 4–10 wk. This limited protection, reminiscent of that observed during the natural history of the disease, does not appear to result from the disappearance of the protective type cells in the recipients, because they persist in the recipients for the length of the experiments (F.L. and M.C.G. manuscript in preparation).
The mechanisms by which CD4+
CD62L+ T cells exert their suppressive activity
are not yet understood, but their phenotype and the cytokines they
produce allow us to draw some conclusions as to the nature of these
cells. Protection against diabetes transfer in NOD mice was recently
shown to be mediated by CD4-
CD8- double negative cells from the thymus of
normal mice (15) or from the spleen of mice carrying a
V14-J281 transgene (16). Both cell populations
include a majority of NK T cells. Peripheral CD4 T cells from wild-type
mice also comprise NK T cells that are CD62L-
(42), thus different from the protective cells described
herein. The cytokine pattern of regulatory cells was shown to influence
the course of the autoimmune process. In different models, Th1 cells
promote disease, whereas Th2 cells prevent Th1 cells from expressing
their pathogenicity. However, in the NOD model there has been no
convincing demonstration that Th2 T cells mediate prevention. For
example, more protective T cell clones derived from unmanipulated NOD
mice belong to the Th1 than to the Th2 subset (43, 44),
and expression of the Th2 cytokine IL-10 in the islets may accelerate
the autoimmune process (45). Moreover, novel types of
regulatory T cells have recently been described mediating suppression
of autoimmune reactions in vitro as well as in vivo. The
immunoregulation provided by the so-called Th3 (46, 47) or
Tr1 (48) subsets is mediated by IL-10 and/or TGF-ß.
Protective clones that share the cytokine profile of Th3 or Tr1 cells
have been isolated from NOD mouse lymphocytes (49, 50).
Recently, CD4+ CD45RBlow
CD38+ T cells were shown to be able to inhibit
both T cell activation and secretion of effector cytokines in vitro by
a mechanism independent of IL-10 and TGF-ß (19). The
spleen CD62L+ regulatory T cells described herein
produced very low levels of IL-2 and IFN- and no IL-4 and IL-10 upon
stimulation in vitro. In addition, anti-cytokine Ab treatment of
mice cotransferred with pathogenic and protective T cells showed that
the protection afforded by spleen CD62L+ T cells
was not mediated by IL-4 and/or IL-10, hence distinct from a Th2- or a
Tr1-type response. Because CD62L+ T cells
produced TGF-ß, it will be interesting to test the role of this
cytokine in prevention of diabetes transfer.
Such regulatory cells have been reproducibly detected in prediabetic
mice from 4 wk of age. In recently diabetic mice,
CD62L+ T cells still represented 
50% of total
T cells and protected against diabetes transfer in two of five
experiments. Altogether, these data suggest that regulatory cells
appear early in life and control the anti-ß cell reaction until
diabetes onset. This is in line with the notion that destruction of the
majority of ß cells is a late and abrupt event in the autoimmune
process (51).
Because the regulatory T cells that are capable of delaying diabetes transfer in NOD-scid hosts are distinct from Th2, Tr1, and NK T cells, they may represent a new regulatory T cell type. The identification of additional phenotypic and functional characteristics of these cells is an important goal for future studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Françoise Lepault, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8603, Hôpital Necker, 161, rue de Sèvres, 75743 Paris, France. E-mail address:
3 Abbreviations used in this paper: NOD, nonobese diabetic; LN, lymph node.
Received for publication June 28, 1999. Accepted for publication October 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+CD4+ thymocytes expressing L-selectin mediate "active tolerance" in the nonobese diabetic mouse. J. Immunol. 161:2620.ß-T cell receptor (TCR)+CD4-CD8- NKT thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187:1047.14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1831.
T cells that prevent murine insulin-dependent diabetes. J. Exp. Med. 184:2167.+ T cells or interferon (IFN)- in a murine model of allergen sensitization. J. Exp. Med. 187:721.This article has been cited by other articles:
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  S. Walters, K. E. Webster, A. Sutherland, S. Gardam, J. Groom, D. Liuwantara, E. Marino, J. Thaxton, A. Weinberg, F. Mackay, et al. Increased CD4+Foxp3+ T Cells in BAFF-Transgenic Mice Suppress T Cell Effector Responses J. Immunol., January 15, 2009; 182(2): 793 - 801. [Abstract] [Full Text] [PDF]
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S. Karumuthil-Melethil, N. Perez, R. Li, and C. Vasu Induction of Innate Immune Response through TLR2 and Dectin 1 Prevents Type 1 Diabetes J. Immunol., December 15, 2008; 181(12): 8323 - 8334. [Abstract] [Full Text] [PDF]
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 X. Huang, D. J. Moore, R. J. Ketchum, C. S. Nunemaker, B. Kovatchev, A. L. McCall, and K. L. Brayman Resolving the Conundrum of Islet Transplantation by Linking Metabolic Dysregulation, Inflammation, and Immune Regulation Endocr. Rev., August 1, 2008; 29(5): 603 - 630. [Abstract] [Full Text] [PDF]
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 D. M. Waid, G. M. Vaitaitis, N. D. Pennock, and D. H. Wagner Jr Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes J. Leukoc. Biol., August 1, 2008; 84(2): 431 - 439. [Abstract] [Full Text] [PDF]
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D. Vallois, M.-C. Gagnerault, P. Avner, U. C. Rogner, C. Boitard, K. Benlagha, A. Herbelin, and F. Lepault Influence of a Non-NK Complex Region of Chromosome 6 on CD4+ Invariant NK T Cell Homeostasis J. Immunol., August 1, 2008; 181(3): 1753 - 1759. [Abstract] [Full Text] [PDF]
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A. L. Tang, J. R. Teijaro, M. N. Njau, S. S. Chandran, A. Azimzadeh, S. G. Nadler, D. M. Rothstein, and D. L. Farber CTLA4 Expression Is an Indicator and Regulator of Steady-State CD4+FoxP3+ T Cell Homeostasis J. Immunol., August 1, 2008; 181(3): 1806 - 1813. [Abstract] [Full Text] [PDF]
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 G. Chen, G. Han, J. Wang, R. Wang, R. Xu, B. Shen, J. Qian, and Y. Li Induction of Active Tolerance and Involvement of CD1d-Restricted Natural Killer T Cells in Anti-CD3 F(ab')2 Treatment-Reversed New-Onset Diabetes in Nonobese Diabetic Mice Am. J. Pathol., April 1, 2008; 172(4): 972 - 979. [Abstract] [Full Text] [PDF]
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K. A. Wong and A. Rodriguez Plasmodium Infection and Endotoxic Shock Induce the Expansion of Regulatory Dendritic Cells J. Immunol., January 15, 2008; 180(2): 716 - 726. [Abstract] [Full Text] [PDF]
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E. N. M. Nolte-'t Hoen, E. P. J. Boot, J. P. A. Wagenaar-Hilbers, J. H. M. van Bilsen, G. J. A. Arkesteijn, G. Storm, L. A. Everse, W. van Eden, and M. H. M. Wauben Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134 J. Leukoc. Biol., January 1, 2008; 83(1): 112 - 121. [Abstract] [Full Text] [PDF]
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S. E. Fineberg, T. T. Kawabata, D. Finco-Kent, R. J. Fountaine, G. L. Finch, and A. S. Krasner Immunological Responses to Exogenous Insulin Endocr. Rev., October 1, 2007; 28(6): 625 - 652. [Abstract] [Full Text] [PDF]
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L. Zhao, L. Sun, H. Wang, H. Ma, G. Liu, and Y. Zhao Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice J. Leukoc. Biol., June 1, 2007; 81(6): 1386 - 1394. [Abstract] [Full Text] [PDF]
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 S. M. Pop, C. P. Wong, Q. He, Y. Wang, M. A. Wallet, K. S. Goudy, and R. Tisch The Type and Frequency of Immunoregulatory CD4+ T-Cells Govern the Efficacy of Antigen-Specific Immunotherapy in Nonobese Diabetic Mice Diabetes, May 1, 2007; 56(5): 1395 - 1402. [Abstract] [Full Text] [PDF]
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S. Makita, T. Kanai, Y. Nemoto, T. Totsuka, R. Okamoto, K. Tsuchiya, M. Yamamoto, H. Kiyono, and M. Watanabe Intestinal Lamina Propria Retaining CD4+CD25+ Regulatory T Cells Is A Suppressive Site of Intestinal Inflammation J. Immunol., April 15, 2007; 178(8): 4937 - 4946. [Abstract] [Full Text] [PDF]
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G. M. Venturi, R. M. Conway, D. A. Steeber, and T. F. Tedder CD25+CD4+ Regulatory T Cell Migration Requires L-Selectin Expression: L-Selectin Transcriptional Regulation Balances Constitutive Receptor Turnover J. Immunol., January 1, 2007; 178(1): 291 - 300. [Abstract] [Full Text] [PDF]
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S. Brode, T. Raine, P. Zaccone, and A. Cooke Cyclophosphamide-Induced Type-1 Diabetes in the NOD Mouse Is Associated with a Reduction of CD4+CD25+Foxp3+ Regulatory T Cells J. Immunol., November 15, 2006; 177(10): 6603 - 6612. [Abstract] [Full Text] [PDF]
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K. J. Scalapino, Q. Tang, J. A. Bluestone, M. L. Bonyhadi, and D. I. Daikh Suppression of Disease in New Zealand Black/New Zealand White Lupus-Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells J. Immunol., August 1, 2006; 177(3): 1451 - 1459. [Abstract] [Full Text] [PDF]
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P. Alard, J. N. Manirarora, S. A. Parnell, J. L. Hudkins, S. L. Clark, and M. M. Kosiewicz Deficiency in NOD Antigen-Presenting Cell Function May Be Responsible for Suboptimal CD4+CD25+ T-Cell-Mediated Regulation and Type 1 Diabetes Development in NOD Mice. Diabetes, July 1, 2006; 55(7): 2098 - 2105. [Abstract] [Full Text] [PDF]
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 B. Valzasina, S. Piconese, C. Guiducci, and M. P. Colombo Tumor-Induced Expansion of Regulatory T Cells by Conversion of CD4+CD25- Lymphocytes Is Thymus and Proliferation Independent. Cancer Res., April 15, 2006; 66(8): 4488 - 4495. [Abstract] [Full Text] [PDF]
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S. E. Weber, J. Harbertson, E. Godebu, G. A. Mros, R. C. Padrick, B. D. Carson, S. F. Ziegler, and L. M. Bradley Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Th1 cells in vivo. J. Immunol., April 15, 2006; 176(8): 4730 - 4739. [Abstract] [Full Text] [PDF]
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U. C. Rogner, F. Lepault, M.-C. Gagnerault, D. Vallois, J. Morin, P. Avner, and C. Boitard The Diabetes Type 1 Locus Idd6 Modulates Activity of CD4+CD25+ Regulatory T-Cells Diabetes, January 1, 2006; 55(1): 186 - 192. [Abstract] [Full Text] [PDF]
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 K. Siegmund, M. Feuerer, C. Siewert, S. Ghani, U. Haubold, A. Dankof, V. Krenn, M. P. Schon, A. Scheffold, J. B. Lowe, et al. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo Blood, November 1, 2005; 106(9): 3097 - 3104. [Abstract] [Full Text] [PDF]
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S. You, M. Belghith, S. Cobbold, M.-A. Alyanakian, C. Gouarin, S. Barriot, C. Garcia, H. Waldmann, J.-F. Bach, and L. Chatenoud Autoimmune Diabetes Onset Results From Qualitative Rather Than Quantitative Age-Dependent Changes in Pathogenic T-Cells Diabetes, May 1, 2005; 54(5): 1415 - 1422. [Abstract] [Full Text] [PDF]
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D. Lundsgaard, T. L. Holm, L. Hornum, and H. Markholst In Vivo Control of Diabetogenic T-Cells by Regulatory CD4+CD25+ T-Cells Expressing Foxp3 Diabetes, April 1, 2005; 54(4): 1040 - 1047. [Abstract] [Full Text] [PDF]
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F. Rharbaoui, D. Bruder, M. Vidakovic, T. Ebensen, J. Buer, and C. A. Guzman Characterization of a B220+ Lymphoid Cell Subpopulation with Immune Modulatory Functions in Nasal-Associated Lymphoid Tissues J. Immunol., February 1, 2005; 174(3): 1317 - 1324. [Abstract] [Full Text] [PDF]
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S. You, C. Chen, W.-H. Lee, T. Brusko, M. Atkinson, and C.-P. Liu Presence of Diabetes-Inhibiting, Glutamic Acid Decarboxylase-Specific, IL-10-Dependent, Regulatory T Cells in Naive Nonobese Diabetic Mice J. Immunol., December 1, 2004; 173(11): 6777 - 6785. [Abstract] [Full Text] [PDF]
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D. Yadav, V. Judkowski, M. Flodstrom-Tullberg, L. Sterling, W. L. Redmond, L. Sherman, and N. Sarvetnick B7-2 (CD86) Controls the Priming of Autoreactive CD4 T Cell Response against Pancreatic Islets J. Immunol., September 15, 2004; 173(6): 3631 - 3639. [Abstract] [Full Text] [PDF]
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N. Duarte, M. Stenstrom, S. Campino, M.-L. Bergman, M. Lundholm, D. Holmberg, and S. L. Cardell Prevention of Diabetes in Nonobese Diabetic Mice Mediated by CD1d-Restricted Nonclassical NKT Cells J. Immunol., September 1, 2004; 173(5): 3112 - 3118. [Abstract] [Full Text] [PDF]
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 Q. Tang, K. J. Henriksen, M. Bi, E. B. Finger, G. Szot, J. Ye, E. L. Masteller, H. McDevitt, M. Bonyhadi, and J. A. Bluestone In Vitro-expanded Antigen-specific Regulatory T Cells Suppress Autoimmune Diabetes J. Exp. Med., June 7, 2004; 199(11): 1455 - 1465. [Abstract] [Full Text] [PDF]
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J. Huehn, K. Siegmund, J. C.U. Lehmann, C. Siewert, U. Haubold, M. Feuerer, G. F. Debes, J. Lauber, O. Frey, G. K. Przybylski, et al. Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells J. Exp. Med., February 2, 2004; 199(3): 303 - 313. [Abstract] [Full Text] [PDF]
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 S. C. Barlow, H. Xu, C. T. Weaver, J. R. Lindsey, T. R. Schoeb, and D. C. Bullard Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes Int. Immunol., February 1, 2004; 16(2): 345 - 351. [Abstract] [Full Text] [PDF]
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 M.-A. Alyanakian, S. You, D. Damotte, C. Gouarin, A. Esling, C. Garcia, S. Havouis, L. Chatenoud, and J.-F. Bach Diversity of regulatory CD4+T cells controlling distinct organ-specific autoimmune diseases PNAS, December 23, 2003; 100(26): 15806 - 15811. [Abstract] [Full Text] [PDF]
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H. Jonuleit and E. Schmitt The Regulatory T Cell Family: Distinct Subsets and their Interrelations J. Immunol., December 15, 2003; 171(12): 6323 - 6327. [Full Text] [PDF]
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S. Gregori, N. Giarratana, S. Smiroldo, and L. Adorini Dynamics of Pathogenic and Suppressor T Cells in Autoimmune Diabetes Development J. Immunol., October 15, 2003; 171(8): 4040 - 4047. [Abstract] [Full Text] [PDF]
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D. Zipris, J.-L. Hillebrands, R. M. Welsh, J. Rozing, J. X. Xie, J. P. Mordes, D. L. Greiner, and A. A. Rossini Infections That Induce Autoimmune Diabetes in BBDR Rats Modulate CD4+CD25+ T Cell Populations J. Immunol., April 1, 2003; 170(7): 3592 - 3602. [Abstract] [Full Text] [PDF]
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V. Szanya, J. Ermann, C. Taylor, C. Holness, and C. G. Fathman The Subpopulation of CD4+CD25+ Splenocytes That Delays Adoptive Transfer of Diabetes Expresses L-Selectin and High Levels of CCR7 J. Immunol., September 1, 2002; 169(5): 2461 - 2465. [Abstract] [Full Text] [PDF]
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M.-C. Gagnerault, J. J. Luan, C. Lotton, and F. Lepault Pancreatic Lymph Nodes Are Required for Priming of {beta} Cell Reactive T Cells in NOD Mice J. Exp. Med., August 5, 2002; 196(3): 369 - 377. [Abstract] [Full Text] [PDF]
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L. Bertry-Coussot, B. Lucas, C. Danel, L. Halbwachs-Mecarelli, J.-F. Bach, L. Chatenoud, and P. Lemarchand Long-Term Reversal of Established Autoimmunity upon Transient Blockade of the LFA-1/Intercellular Adhesion Molecule-1 Pathway J. Immunol., April 1, 2002; 168(7): 3641 - 3648. [Abstract] [Full Text] [PDF]
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R. H. Friedline, C. P. Wong, D. A. Steeber, T. F. Tedder, and R. Tisch L-Selectin Is Not Required for T Cell-Mediated Autoimmune Diabetes J. Immunol., March 15, 2002; 168(6): 2659 - 2666. [Abstract] [Full Text] [PDF]
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Y. Bai, J. Liu, Y. Wang, S. Honig, L. Qin, P. Boros, and J. S. Bromberg L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance J. Immunol., February 15, 2002; 168(4): 1579 - 1589. [Abstract] [Full Text] [PDF]
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M. G. von Herrath, B. Coon, T. Wolfe, and L. Chatenoud Nonmitogenic CD3 Antibody Reverses Virally Induced (Rat Insulin Promoter-Lymphocytic Choriomeningitis Virus) Autoimmune Diabetes Without Impeding Viral Clearance J. Immunol., January 15, 2002; 168(2): 933 - 941. [Abstract] [Full Text] [PDF]
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K. Jooss, B. Gjata, O. Danos, H. von Boehmer, and A. Sarukhan Regulatory function of in vivo anergized CD4+ T cells PNAS, June 28, 2001; (2001) 151088898. [Abstract] [Full Text] [PDF]
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A. M. Yamamoto, Y. Chernajovsky, F. Lepault, O. Podhajcer, M. Feldmann, J.-F. Bach, and L. Chatenoud The Activity of Immunoregulatory T Cells Mediating Active Tolerance Is Potentiated in Nonobese Diabetic Mice by an IL-4-Based Retroviral Gene Therapy J. Immunol., April 15, 2001; 166(8): 4973 - 4980. [Abstract] [Full Text] [PDF]
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C. Carnaud, J.-M. Gombert, O. Donnars, H.-J. Garchon, and A. Herbelin Protection Against Diabetes and Improved NK/NKT Cell Performance in NOD.NK1.1 Mice Congenic at the NK Complex J. Immunol., February 15, 2001; 166(4): 2404 - 2411. [Abstract] [Full Text] [PDF]
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J. Salaun, N. Simmenauer, P. Belo, A. Coutinho, and N. M. Le Douarin Grafts of supplementary thymuses injected with allogeneic pancreatic islets protect nonobese diabetic mice against diabetes PNAS, January 22, 2002; 99(2): 874 - 877. [Abstract] [Full Text] [PDF]
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K. Jooss, B. Gjata, O. Danos, H. von Boehmer, and A. Sarukhan Regulatory function of in vivo anergized CD4+ T cells PNAS, July 17, 2001; 98(15): 8738 - 8743. [Abstract] [Full Text] [PDF]
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, n =
5) or mixed with 4 x 106 CD62L- T cells
(
, n = 4), 15 x 106
CD62L+ T cell equivalents (
, n = 5),
or 15 x 106 purified CD62L+ T cells (
,
n = 6).
,
n = 5), 5 x 106 (
,
n = 5), or 20 x 106 (
, n = 6) 15 x
106 CD4+ (x, n
= 6), 15 x 106 CD62+ T cells (
