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ß+CD4+ Thymocytes Expressing L-Selectin Mediate "Active Tolerance" in the Nonobese Diabetic Mouse1
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Institut National de la Santé et de la Recherche Médicale, Unité 25, and
Centre National de la Recherche Scientifique, Unité de Recherche Associée 1461, Hôpital Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+CD8- thymocytes from
prediabetic NOD mice also express this inhibitory capacity. Selection
using an L-selectin (CD62L)-specific Ab showed that
TCRß+CD4+CD62L+ thymocytes,
emerging from the mainstream differentiation pathway, concentrate this
ability to regulate autoreactive effectors. Compared with mature
TCRß+CD8- thymocytes, significantly lower
numbers of TCRß+CD4+CD62L+
were sufficient to achieve an efficient inhibition of disease transfer
into NOD-scid recipients. This protective ability was
potentiated following in vitro culture in the presence of IL-7. In
contrast, TCRß+CD62L- thymocytes, highly
enriched in class I-restricted NK T cells, were unable to influence
diabetes transfer. Identical results were obtained using thymocytes
that have been cultured in vitro for 4 days in the presence of IL-7.
These results support the active role in NOD mice of a thymus-derived
CD4+ subset that controls peripheral pathogenic autoimmune
effectors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing Th1
subset, and CD8+ T cells have been implicated in the
pathogenesis of IDDM (3, 4, 5, 6, 7, 8). Diabetogenic T cells, which transfer acute
IDDM into immunoincompetent syngeneic recipients, are present in high
frequency in the spleens of diabetic NOD mice (3, 9). In parallel to
these effector cells, there is substantial evidence showing the
presence, especially in young prediabetic NOD mice, of a subset of T
lymphocytes mediating "active tolerance," that is, exerting active
control of or a down-regulatory effect on diabetogenic lymphocytes or
their precursors. Thus, cotransfer experiments have shown that
CD4+ T splenocytes from prediabetic animals fully prevent
the transfer of disease by diabetogenic cells (10, 11). Moreover,
diabetogenic T cells can transfer IDDM into syngeneic adult mice only
if they are sublethally irradiated (2, 9, 10, 12). Effective depletion
of CD4+ lymphocytes, as obtained after adult thymectomy and
treatment with specific Abs, can effectively substitute for the
irradiation (13). This further corroborates the CD4 nature of the
protective cell. Lastly, cyclophosphamide, an alkylating agent that in
different models, including delayed-type hypersensitivity (14) and
experimental allergic encephalomyelitis (15, 16, 17, 18), has been shown to
selectively affect T cell-dependent regulation, is able to trigger
acute diabetes within 2 wk when injected into young prediabetic NOD
mice (2, 12, 19). A mere toxic effect on ß cells has been excluded by
the work of Charlton et al., which showed that cyclophosphamide-induced
IDDM is prevented by the transfusion of mononuclear cells from
nondiabetic NOD mice (19). These regulatory T cells have been evidenced not only in peripheral lymphoid organs but also in the thymus. Boitard et al. reported that, when cotransferred with diabetogenic T cells, total thymocytes from young prediabetic NOD mice successfully protected from disease as efficiently as did their spleen CD4+ cells (10). Moreover, Dardenne et al. showed that thymectomy performed in NOD females at weaning (3 wk of age) significantly increased the incidence of spontaneous IDDM, thus confirming the thymic origin of the regulatory population (20).
The aim of the present work has been to further characterize the phenotypic and functional properties of the thymic population capable of down-regulating diabetogenic effectors. In this vein we have used a series of T cell markers that dissect functionally distinct subsets of thymocytes (21). This issue is of particular interest given the recent data from our group (21) and from that of Baxter et al. (22) showing that NOD mice exhibit a marked deficiency in the recently described NK T cell subset (23, 24, 25, 26, 27). Based on the evidence that this subset produces massive amounts of IL-4 upon TCR cross-linking, one major question was to determine whether NK T cells could directly modulate in conventional cotransfer experiments the pathogenic ability of diabetogenic T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD (Kd, I-Ag7, Db) and
NOD-scid mice were bred in our animal facilities under
specific pathogen-free conditions; in females, spontaneous IDDM appears
by 14 wk of age (80% incidence at 30 wk of age) and is preceded by
insulitis at 4 to 6 wk. Colorimetric strips were used to monitor
glycosuria (Glukotest, Boehringer Mannheim, Mannheim, Germany) and
glycemia (Haemoglukotest and Reflolux F, Boehringer Mannheim). Mice
were screened for glycosuria twice a week. When glycosuric the animals
were also screened for hyperglycemia. Mice were considered diabetic
when glycemia 
3 g/liter was scored at two consecutive measurements.
Abs and FACS analysis
Abs to CD4 (clone GK1.5), to CD8 (clone 53.6.7 and clone 3-155),
to ßTCR (clone H57-597), to Vß8TCR (clone F23.1), and to HSA
(anti-CD24, clone J11d) were purified and fluoresceinated and/or
biotinylated in our laboratory. PE-anti-CD4 (clone RM4.5),
PE-anti-CD24 (clone M1/69), FITC- and biotin-anti-CD44 (clone 1
M7.8), biotin-anti-CD62L (MEL-14), PE-anti-HSA (clone M.1/69),
and FITC-anti-CD8 (clone 53.6.7) were obtained from PharMingen (San
Diego, CA). Biotin-anti-3G11 mAb was a gift from Dr. A. Bendelac
(Department of Molecular Biology, Princeton University, Princeton, NJ).
Surface markers were assessed by flow cytometry. Briefly, cells were
stained in HBSS containing 5% heat-inactivated FCS (Techgen, Les Ulis,
France) and 0.01% sodium azide and were incubated for 30 min with the
appropriate concentration of biotin-labeled mAbs in 96-well
round-bottom microplates on ice. After three washes, the cells were
incubated for another 30 min with FITC-labeled mAbs and PE-labeled
streptavidin or PE-labeled mAbs. For triple staining, during the second
step, streptavidin-Tricolor (Caltag) was added to the appropriate FITC-
and PE-labeled mAb combination. Control stainings were performed using
isotype-matched, biotinylated FITC- or PE-labeled irrelevant Abs. Flow
cytometry was performed on a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA). A minimum of 1 x 104 events were
acquired on a gate including viable lymphocytes. For acquisition and
analysis, the software used was LYSYS II or, in more recent
experiments, CellQuest (Becton Dickinson).
Cell purifications
Thymi and spleens were carefully recovered from exsanguinated mice. For cell purification experiments, pools of 5 to 15 thymi or spleens were used. Mature double-negative and CD4+ thymocytes were enriched by means of complement depletion. Whole thymocyte suspensions were incubated at 37°C for 40 min with the IgM Abs 3-155 (rat anti-mouse CD8) and J11d (rat anti-mouse heat stable Ag, anti-HSA) plus complement (Low-Tox Rabbit Complement, Cedarlane, Ontario, Canada). Mature CD8+, immature CD4+CD8+, and the vast majority of immature HSA+CD4-CD8- thymocytes were thus eliminated. Viable cell suspensions were recovered after density gradient centrifugation (J. Prep., Techgen, Les Ulis, France; 1400 rpm for 20 min). The cells recovered at the interface were washed twice in culture medium. To assess the depletion, HSA-CD8- thymocytes were stained with a combination of HSA and CD8 Abs different from those used for the depletion (clones M.1/69 and clone 53.6.7, respectively). The depleted population obtained contained >95% HSA-CD8- cells as assessed by flow cytometry.
When needed, HSA-CD8- thymocytes were further
subdivided on the basis of CD62L and 3G11 expression using magnetic
activated cell sorting (MACS, Miltenyi Biotech, Bergisch-Gladbach,
Germany) as previously described (21). Briefly,
HSA-CD8- thymocytes were incubated with the
appropriate concentration of anti-CD62L and anti-3G11 mAbs. The
cells were washed and incubated with the streptavidin-coated
paramagnetic beads (Miltenyi Biotech) and passed through the column
within the magnetic activated cell sorting device according to the
manufacturer’s instructions. The purity of the CD62L 3G11-negative and
-positive fractions recovered was analyzed by flow cytometry after
staining with PE-labeled streptavidin. The purity of the sorted cells
was in all cases >90% (see Fig. 1
A), and recovery ranged from 50
to 70%.
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In vitro cultures and cytokine production
Cells were cultured in RPMI 1640 culture medium (Life Technologies, Gaithersburg, MD) supplemented with Glutamax, 10% FCS (Techgen), 0.05 mM ß-ME, penicillin (100 IU/ml), and streptomycin (100 µg/ml). When needed, thymocyte suspensions were cultured for 60 h in the presence of human rIL-7 according to the method we previously described (29, 30). Briefly, total thymocyte suspensions (20 x 106/well) were cultured in 24-well plastic plates (Costar, Cambridge, MA) in complete medium supplemented with rIL-7 (1000 U/ml; Sanofi, Labege, France). After 60 h of culture at 37°C in a humidified atmosphere containing 10% CO2, viable cells were recovered by centrifugation (1400 rpm for 20 min) on a density gradient (J. Prep., Techgen). The cells at the interface were recovered, washed twice in complete medium, and used for in vitro (phenotype analysis and cytokine production) and in vivo assays. When needed, they were also used in magnetic sorting experiments (described above).
To assess cytokine production, lymphocyte suspensions were plated in
triplicate (1–2 x 105/well; 200 µl final volume)
in 96-well round-bottom microplates (Nunc, Roskilde, Denmark) coated
with 10 µg/ml of anti-ßTCR (H57-597). Supernatants were
recovered at 48 h of culture at 37°C in a humidified atmosphere
containing 5% CO2 and stored at -80°C until tested. In
control cultures coating with the ßTCR mAb was omitted, and no
cytokines were detected in the supernatants.
IL-4 and IFN- in the supernatants were measured by means of two-site
sandwich ELISAs, as previously described (21). The 11B11
(anti-IL-4) and AN18 (anti-IFN-) mAbs were used for coating
the plates (capture mAbs), and biotinylated-BVD6 (anti-IL-4) and
R46A2 (anti-IFN-) mAbs were used as second Abs. The 11B11
hybridoma was provided by W. Paul (National Institutes of Health,
Bethesda, MD), and the BVD6, AN18 hybridomas were provided by A.
O’Garra (DNAX, Palo Alto, CA). Briefly, the plates were coated
overnight with the capture mAbs diluted in carbonate-bicarbonate buffer
(pH 9.8; 0.1 M). After washing (PBS/0.1% Tween) and blocking (PBS/1%
BSA), the test samples and the standards were distributed, and the
plates were incubated for 2 h at room temperature. Following
incubation with the biotinylated mAbs and subsequently with horseradish
peroxidase-labeled streptavidin (Vector, Burlingame, CA), the plates
were washed, and ortho-phenylene-diamine was used as a substrate
(Sigma, St. Louis, MO). The cytokine concentrations were expressed as
nanograms per milliliter, based on a calibration curve established for
each assay using serial dilutions of recombinant standards. Mouse rIL-4
and IFN- were obtained from R&D (Minneapolis, MN). The sensitivities
of the IL-4 and IFN- assays were 0.2 and 0.1 ng/ml, respectively.
Adoptive cell transfer
Depending on the experiment, two sorts of recipients were used, adult irradiated NOD males or, in more recent experiments, NOD-scid. As already reported by several groups, including ours, adult 6- to 8-wk-old NOD males have to be sublethally irradiated (750 rad) to be used as recipients of cell transfers (9, 10). In such recipients the various transferred cells were injected 24 h following the irradiation according to our conventional protocol (10). Depending on the experiment, the animals received either a single cell population or, in the case of cotransfer experiments, a mixture of two distinct cell populations. The precise cell numbers used varied depending on the experiments and are detailed in Results.
In all the experiments, diabetogenic cells were recovered by gentle disruption from the spleen of overtly diabetic NOD females. Pools of 7 to 10 diabetic mouse spleens were used. It has been well established by several laboratories, including ours, that diabetes transfer is exclusively mediated by T cells (3, 9, 31, 32). Thus, the numbers of diabetogenic spleen cells to inject were deduced according to the proportions of CD3+ cells, as assessed by FACS analysis, scored in each pooled spleen cell preparation. This is referred to in the text as T cell equivalents.
Other transferred populations included whole thymocyte suspensions and purified thymocyte subsets recovered from either fresh thymocyte suspensions or thymocytes recovered after a 60-h in vitro culture in the presence of IL-7.
Histopathology
Sections of paraffin-embedded or frozen pancreata were used. For conventional histopathology, sections were stained with hematoxylin and eosin to score mononuclear cell infiltration as follows: grade 0 = normal islets, grade 1 = focal or peripheral insulitis (lymphocytes surrounding the islet, but no destruction of endocrine cells as assessed by labeling with anti-insulin Abs), and grade 2 = invasive destructive insulitis.
Statistical analysis
When appropriate, results were analyzed using the
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously shown that total thymic cells
collected from young prediabetic NOD mice could protect from the
transfer of diabetes in adult irradiated recipients (10). The present
experiments confirmed these results and extended them to
NOD-scid recipients. As shown in Table I, in both adult irradiated
recipients and NOD-scid mice 50 x
106 total thymocytes from NOD females fully inhibited the
diabetogenic capacity of 5 x 106 spleen T cell
equivalents (see Materials and Methods) from diabetic NOD
mice. This protective effect was dose dependent, since 5 x
106 thymocytes failed to impede diabetes development.
Protector cells were collected from both young (3 wk) and adult
(8-wk-old) prediabetic animals. In all cases the thymocyte population
transferred included 80 to 85% of double-positive
CD4+CD8+ cells and 15 to 20% of mature
single-positive and double-negative cells.
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Subsequently, experiments were designed to determine whether the
thymocyte population endowed with the protective ability belonged to
the immature or mature thymic compartment. Complement depletion
experiments were thus performed using an Ab to the heat-stable Ag,
which is expressed on immature double-positive thymocytes. As detailed
in Table II, the mature HSA-
population reproducibly retained the protective activity; as few as
7.5 x 106 HSA- thymocytes were
sufficient to abolish the pathogenic capacity of 5 x
106 diabetogenic T cell equivalents. CD8 single-positive
thymocytes were not essential for this effect, since an additional
complement-mediated depletion with an Ab to CD8 did not modify the
protective capacity of HSA- thymocytes (Tables II and
III). These data also argue against the fact
that the protection afforded by 50 x 106, but not
5 x 106, thymocytes could be due to a dilution effect
of the diabetogenic population.
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, HSA+ thymocytes cells were totally incapable
at transferring protection.
As for total thymocytes, the protective ability was evidenced with
purified mature thymocytes collected not only at 3 wk but also at 9 to
10 wk of age (Table III).
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There is extensive evidence in the literature that mature
HSA-CD8- thymocytes constitute a
heterogeneous population. One may distinguish within this population
mainstream TCRß+CD4+ cells from the
discrete subset of recently characterized thymocytes sharing NK and
TCRs, namely NK T cells (23, 24, 25, 26, 27). A major characteristic that
distinguishes these two subsets is their differential expression of
membrane receptors such as CD44, 3G11, and CD62L (21, 24, 25, 26, 27, 33, 34, 35, 36).
As we previously reported (21), 60 to 70% of
HSA-CD8- cells are CD62L+ and
correspond to the mainstream population that exclusively includes the
CD4+ single-positive thymocytes (Fig. 1A). In
contrast, the CD62L- population that, depending on the age
of the thymus donors, comprises 15 to 20% of the cells, includes both
double-negative and CD4+ single-positive thymocytes showing
an intermediate ßTCR expression typical of NK T cells. Thymic NK T
cells, as opposed to CD4+ mainstream thymocytes, also lack
the 3G11 marker (21, 34) (data not shown).
Thus, in further immunomagnetic cell sorting experiments we used the
L-selectin receptor and the 3G11 marker that are expressed at high
density on mainstream single-positive CD4+ but not on NK T
thymocytes. One representative experiment is detailed in Figure 1 to
show the phenotypic distribution of CD62L and 3G11 before and after
sorting. As we previously reported (21), this purification method
allows high enrichment of the CD62L-3G11-
NK-like T subset that concentrates the capacity to rapidly produce high
amounts of IL-4 upon TCR cross-linking. This latter functional capacity
is exclusively attributed to NK T cells in the thymus (21, 24, 26, 27, 29).
The present results reproducibly showed that it is the
CD4+CD62L+3G11+ and not the
CD62L-3G11- population (the subset that
includes NK-like T cells (Fig. 1A) that retains the
protective ability of HSA-CD8- cells (Table III). To maintain, as much as possible, homogeneous experimental
conditions, the absolute numbers of sorted cells used for cotransfer
experiments were deduced from the proportion of the subset under
analysis in the original HSA-CD8- population.
The cytokine-producing ability (i.e., IL-4 and IFN-) upon TCR
cross-linking of the various cell populations cotransferred is detailed
in Figure 1B. The
CD4+CD62L+3G11+ thymocyte subset
that concentrated the protective ability was, as expected, a poor IL-4
producer compared with the CD62L-3G11-
NK-like T cell-enriched subset.
IL-7 in vitro treatment potentiates the protective capacity of mature L-selectin-positive thymocytes
We have previously reported that, upon in vitro culture in the
presence of IL-7, mature thymocytes significantly expand (29, 30). The
population recovered after 60 h of culture in the presence of IL-7
exclusively included mature single-positive or double-negative T cells,
with a significant proportion of NK-like T cells within the
CD62L-3G11- subset (29, 30). We explored
whether the in vitro IL-7 treatment could modulate the protective
capacity of mature thymocytes. In a first series of experiments total
thymocytes were incubated with IL-7, using already described
experimental conditions (29, 30), namely, 60 h of culture at high
cell density (20 x 106/well) in the presence of 1000
U/ml of human rIL-7. The distribution of
CD62L+3G11+ among ßTCR+ and
Vß8+ thymocytes before and after IL-7 culture is shown in
Figure 2.
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) or
NOD-scid mice (data not shown). By comparison, 50 x
106 total thymocytes (including about 10–15% mature
cells) or 5 x 106 purified
HSA-CD8- thymocytes were needed. The same
results were obtained when purified mature
HSA-CD8- thymocytes, instead of total
thymocytes, were used for the IL-7 cultures (Table IV). Here again, as
few as 2 x 106 cultured cells very effectively
protected from disease transfer.
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A). At variance,
CD62L-3G11- cells that include
CD4- and CD4-CD8- NK-like T
thymocytes were totally ineffective in conferring protection (Fig. 3A). As detailed in Figure 3B, a majority of the
islets from the pancreas of protected animals showed a peripheral
noninvasive/nondestructive insulitis.
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) upon TCR
cross-linking of the various IL-7 cultured cell populations used in
cotransfers is detailed in Table V. The
TCRß+CD62L+3G11+ thymocyte
subset that concentrated the protective ability remained, a poor IL-4
producer compared with the CD62L-3G11-
NK-like T cell-enriched subset. It is interesting to emphasize that
when cultured in the presence of IL-7,
CD62L+3G11+ thymocytes acquire the capacity to
produce massive amounts of IFN-. Indeed, the values were as high as
those measured in the supernatants from stimulated cultures of
TCRß+CD62L-3G11- cells that
are highly enriched in NK-like T thymocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunopathologic significance of these protective cells is still uncertain. It is tempting, however, to relate them to the resistance to diabetes transfer that develops in NOD mice by 3 wk of age (12), the acceleration of diabetes onset after thymectomy at this same age (i.e., weaning) (20), and the induction of acute diabetes upon cyclophosphamide treatment (19, 38). The main question is then to characterize more precisely the nature of these regulatory cells that are not only found in the thymus, but also in the periphery, i.e., the spleen (F. Lepault, unpublished observations). Indirect data have suggested that they could express a preferential Th2 phenotype. Thus, NOD mice are kept disease free by systemic treatments with IL-4 or IL-10 (39, 40, 41), and a Th1/Th2 shift is evidenced in mice protected from disease by different means, including the administration of ß cell autoantigens (2, 42, 43, 44), the injection of CFA or Calmette-Guérin bacillum (2, 45), or the islet-directed transgenic expression of a mutated I-Ag7 (46, 47). However, more direct evidence is lacking, since Th2 cell lines or clones cannot protect from disease transfer by diabetogenic cells (7, 8). Recent data have suggested a major role for TGF-ß in this protection. Thus, TGF-ß-producing clones are protective in cotransfer, and an Ab to TGF-ß is able to break the tolerance induced upon oral insulin administration (48, 49).
Mature CD8- thymocytes constitute a heterogeneous
population including TCRß+ cells derived from both the
mainstream and the nonmainstream differentiation pathways. Among
nonmainstream thymocytes, a discrete subset was recently described that
expresses receptors specific of both T cells (TCR) and NK cells (NK1.1,
Ly 49), hence termed NK T cells. NK T cells include CD4+
single-positive as well as double-negative cells, which share an
unusually restricted V14 Vß8 TCR repertoire. Major additional
distinctive features are, first, their restriction by nonpolymorphic
MHC class I molecules, CD1 being one major ligand (23, 24, 25, 26, 27), and second,
their expression of an activated cell phenotype, CD44+,
CD62L- (L-selectin, MEL-14-),
3G11-, CD122+ (the ß-chain of the IL-2
receptor). Lastly, upon TCR cross-linking, NK T cells produce massive
amounts of different cytokines, such as IL-4 and IFN- (23, 24, 25, 26, 50).
Some authors have suggested that, through the production of IL-4, NK T
cells promote Th2-type responses. This was supported by the data
showing the absence of IgE production following anti-IgD polyclonal
activation in ß2m KO mice that totally lack NK T cells
(51). However, the uniqueness of this effect has been recently
questioned, since in the same experimental conditions CD1 knockout
mice, which also lack NK T cells, can mount IgE responses (52).
Although the NOD strain does not express the NK1.1 allele, NK T cells
can be reliably detected using a combination of Abs to CD44, CD62L,
3G11, and Vß8. Using this approach, we could verify in the thymus of
normal C57BL/6 mice that the
ßTCR+CD44+CD62L-3G11-
subset was NK1.1 positive and concentrated the ability to rapidly
produce IL-4 upon TCR cross-linking (21). We showed a clear-cut deficit
in the numbers of NK T thymocytes in 3- and 8-wk-old prediabetic NOD
mice (21). This quantitative abnormality correlated with a major defect
in the IL-4-producing capacity of NOD thymocytes (21, 39). Concordant
data have been published by Baxter et al., who showed that
CD4-CD8 cells are significantly decreased in both the
thymus and the periphery of NOD mice (22).
It was thus relevant to directly assess the cotransfer behavior of
purified mainstream- vs nonmainstream (NK T)-derived thymocytes. To
address this question we took advantage of the absence of L-selectin on
NK T cells contrasting with its presence in a large proportion of
mature thymocytes (21, 25, 26, 27). Mature NOD
CD4-CD8- thymocytes were further selected by
means of immunomagnetic cell sorting using MEL-14 Ab. The results
clearly indicated that the TCRß+ thymocytes capable of
protecting in cotransfer were CD62L+ or, alternatively,
depended on an TCRß+CD62L+ subset to exert
their protective ability. The frequency and the kinetics of diabetes
transfer in recipients coinjected with
TCRß+CD62L- NK-like T thymocytes were
identical with those seen when injecting diabetogenic cells alone.
These results apparently conflict with those recently reported by
Baxter et al. (22). However, these authors used a totally different
strategy and concentrated on whole unseparated double-negative
thymocytes, which include both mainstream (CD62L+) and
nonmainstream NK-like T thymocytes (CD62L-). The injection
of these double-negative thymocytes into 4-wk-old diabetes-prone
unmanipulated NOD recipients fully prevented the development of the
spontaneous disease (22). On the basis of our present results we would
tend to ascribe this protective capacity to the
CD62L+CD4-CD8- subset that is
known to include the precursors of the
TCRß+CD62L+CD4+ subset.
This conclusion is also in keeping with the fact that in the periphery, i.e., the spleen of adult NOD mice, CD4+ protective cells also express high levels of L-selectin (F. Lepault, manuscript in preparation) while diabetogenic T splenocytes concentrate among CD62L- (53). These data are also concordant with those showing no or very low levels of L-selectin among intraislet T lymphocytes despite the expression of vascular addressins on the postcapillary venules of inflamed NOD islets (54). Thus, CD62L, initially described as a homing receptor, that is, an addressin ligand (55), seems to provide a unique marker to separate, in both the thymus and the periphery, effector from regulatory cells mediating active tolerance.
Interestingly, this is also the case in PVG.RT1u rats
that, following adult thymectomy and repeated low-dose irradiation,
develop autoimmune T cell-mediated IDDM. In these animals disease is
effectively prevented by the transfer of
TCRß+CD4+RT6+CD45RClowCD62L+
cells (56, 57, 58, 59). Although upon activation, these cells exhibited a
preferential Th2 phenotype (producing IL-4 but not IFN-) a
neutralizing Ab to rat IL-4 was unable to reproducibly abrogate the
protection. In this context, it is interesting to note that in our NOD
model and confirming our previously published results (21), upon
activation by means of TCR cross-linking the protective
TCRß+CD62L+ thymocyte subset produced
minimal amounts of IL-4 compared with the nonprotective
TCRß+CD62L- NK T subset.
Although the importance of CD4+CD62L+ T cells in the active tolerance that protects young NOD mice from diabetes, their relationship with the NK T subset remains obscure. It is tempting to see a relationship between the early NK T cell defect consistently found in NOD mice and the failure of the regulatory/protector cells concomitant to diabetes onset. However, the present results argue against a direct role of NK T cells in protection, since they can be physically separated from the subset inhibiting diabetes transfer. It is also difficult to see them as precursors of the protective population, since, at 3 wk of age, the NOD thymus contains a large amount of protector cells and yet is very deficient in NK T cells. More likely would be the involvement of NK T cells in maintenance or long term homeostasis of the protector cells.
One crucial, still unsolved issue concerns the specificity as well as the pathways driving the intrathymic generation of the regulatory T cells described in the various models discussed above. In a series of very elegant experiments, the groups of Le Douarin and Coutinho have demonstrated, initially in birds (quail to chick grafts) and subsequently in mice, that the thymic epithelium could actively select for cells mediating active tolerance (60, 61). It is tempting to relate these observations to various reports showing the expression of different autoantigens within the thymus (59).
To conclude, the NOD mouse is protected from disease onset during the first 2 or 3 mo of life by CD4 regulatory T cells expressing the CD62L+ phenotype. Interestingly enough, the thymus-derived CD4+ subset identified has the potential to control peripheral, fully differentiated pathogenic autoimmune effectors. Although these CD4 T cells are physically distinct from NK-like T cells, a relationship between these two populations cannot at this point be formally excluded.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lucienne Chatenoud, Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, 161 rue de Sèvres, 75743 Paris, France.
3 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; HSA, heat-stable antigen; PE, phycoerythrin.
Received for publication January 16, 1998. Accepted for publication April 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. upon activation by anti-CD3 or CD1. J. Immunol. 159:2240.This article has been cited by other articles:
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  A. P.R. Sutherland, T. Van Belle, A. L. Wurster, A. Suto, M. Michaud, D. Zhang, M. J. Grusby, and M. von Herrath Interleukin-21 Is Required for the Development of Type 1 Diabetes in NOD Mice Diabetes, May 1, 2009; 58(5): 1144 - 1155. [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. 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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 S. Steffens, F. Burger, G. Pelli, Y. Dean, G. Elson, M. Kosco-Vilbois, L. Chatenoud, and F. Mach Short-Term Treatment With Anti-CD3 Antibody Reduces the Development and Progression of Atherosclerosis in Mice Circulation, October 31, 2006; 114(18): 1977 - 1984. [Abstract] [Full Text] [PDF]
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J. Harnaha, J. Machen, M. Wright, R. Lakomy, A. Styche, M. Trucco, S. Makaroun, and N. Giannoukakis Interleukin-7 Is a Survival Factor for CD4+ CD25+ T-Cells and Is Expressed by Diabetes-Suppressive Dendritic Cells Diabetes, January 1, 2006; 55(1): 158 - 170. [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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 S. M. Pop, C. P. Wong, D. A. Culton, S. H. Clarke, and R. Tisch Single cell analysis shows decreasing FoxP3 and TGF{beta}1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes J. Exp. Med., April 18, 2005; 201(8): 1333 - 1346. [Abstract] [Full Text] [PDF]
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 S. You, G. Slehoffer, S. Barriot, J.-F. Bach, and L. Chatenoud Unique role of CD4+CD62L+ regulatory T cells in the control of autoimmune diabetes in T cell receptor transgenic mice PNAS, October 5, 2004; 101(suppl_2): 14580 - 14585. [Abstract] [Full Text] [PDF]
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J. A. Bluestone and Q. Tang Therapeutic vaccination using CD4+CD25+ antigen-specific regulatory T cells PNAS, October 5, 2004; 101(suppl_2): 14622 - 14626. [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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C. Vasu, B. S. Prabhakar, and M. J. Holterman Targeted CTLA-4 Engagement Induces CD4+CD25+CTLA-4high T Regulatory Cells with Target (Allo)antigen Specificity J. Immunol., August 15, 2004; 173(4): 2866 - 2876. [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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 L. A. Stephens, A. N. Barclay, and D. Mason Phenotypic characterization of regulatory CD4+CD25+ T cells in rats Int. Immunol., February 1, 2004; 16(2): 365 - 375. [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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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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R. D. Molano, A. Pileggi, T. Berney, R. Poggioli, E. Zahr, R. Oliver, C. Ricordi, D. M. Rothstein, G. P. Basadonna, and L. Inverardi Prolonged Islet Allograft Survival in Diabetic NOD Mice by Targeting CD45RB and CD154 Diabetes, April 1, 2003; 52(4): 957 - 964. [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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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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B. Wang, Y.-B. Geng, and C.-R. Wang Cd1-Restricted Nk T Cells Protect Nonobese Diabetic Mice from Developing Diabetes J. Exp. Med., August 6, 2001; 194(3): 313 - 320. [Abstract] [Full Text] [PDF]
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L. D. Poulton, M. J. Smyth, C. G. Hawke, P. Silveira, D. Shepherd, O. V. Naidenko, D. I. Godfrey, and A. G. Baxter Cytometric and functional analyses of NK and NKT cell deficiencies in NOD mice Int. Immunol., July 1, 2001; 13(7): 887 - 896. [Abstract] [Full Text] [PDF]
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R. Tisch, B. Wang, M. A. Atkinson, D. V. Serreze, and R. Friedline A Glutamic Acid Decarboxylase 65-Specific Th2 Cell Clone Immunoregulates Autoimmune Diabetes in Nonobese Diabetic Mice J. Immunol., June 1, 2001; 166(11): 6925 - 6936. [Abstract] [Full Text] [PDF]
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 H.-R. Jiang, N. Taylor, L. Duncan, A. D Dick, and J. V Forrester Total dose and frequency of administration critically affect success of nasal mucosal tolerance induction Br J Ophthalmol, June 1, 2001; 85(6): 739 - 744. [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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Y. Kuniyasu, T. Takahashi, M. Itoh, J. Shimizu, G. Toda, and S. Sakaguchi Naturally anergic and suppressive CD25+CD4+ T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation Int. Immunol., August 1, 2000; 12(8): 1145 - 1155. [Abstract] [Full Text] [PDF]
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D. Olivares-Villagomez, A. K. Wensky, Y. Wang, and J. J. Lafaille Repertoire Requirements of CD4+ T Cells That Prevent Spontaneous Autoimmune Encephalomyelitis J. Immunol., May 15, 2000; 164(10): 5499 - 5507. [Abstract] [Full Text] [PDF]
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O. Annacker, O. Burlen-Defranoux, R. Pimenta-Araujo, A. Cumano, and A. Bandeira Regulatory CD4 T Cells Control the Size of the Peripheral Activated/Memory CD4 T Cell Compartment J. Immunol., April 1, 2000; 164(7): 3573 - 3580. [Abstract] [Full Text] [PDF]
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F. Lepault and M. C. Gagnerault Characterization of Peripheral Regulatory CD4+ T Cells That Prevent Diabetes Onset in Nonobese Diabetic Mice J. Immunol., January 1, 2000; 164(1): 240 - 247. [Abstract] [Full Text] [PDF]
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M. Itoh, T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, and S. Sakaguchi Thymus and Autoimmunity: Production of CD25+CD4+ Naturally Anergic and Suppressive T Cells as a Key Function of the Thymus in Maintaining Immunologic Self-Tolerance J. Immunol., May 1, 1999; 162(9): 5317 - 5326. [Abstract] [Full Text] [PDF]
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