By
From the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
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Abstract |
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Previous studies have shown that induction of autoimmune diabetes by adult thymectomy and
split dose irradiation of PVG.RT1u rats can be prevented by their reconstitution with peripheral CD4+CD45RCTCR-
/
+RT6+ cells and CD4+CD8
thymocytes from normal syngeneic donors. These data provide evidence for the role of regulatory T cells in the prevention
of a tissue-specific autoimmune disease but the mode of action of these cells has not been reported previously. In this study, autoimmune thyroiditis was induced in PVG.RT1c rats using a
similar protocol of thymectomy and irradiation. Although a cell-mediated mechanism has been
implicated in the pathogenesis of diabetes in PVG.RT1u rats, development of thyroiditis is independent of CD8+ T cells and is characterized by high titers of immunoglobulin (Ig)G1 antithyroglobulin antibodies, indicating a major humoral component in the pathogenesis of disease. As with autoimmune diabetes in PVG.RT1u rats, development of thyroiditis was
prevented by the transfer of CD4+CD45RC
and CD4+CD8
thymocytes from normal donors but not by CD4+CD45RC+ peripheral T cells. We now show that transforming growth
factor (TGF)-
and interleukin (IL)-4 both play essential roles in the mechanism of this protection since administration of monoclonal antibodies that block the biological activity of either of
these cytokines abrogates the protective effect of the donor cells in the recipient rats. The prevention of both diabetes and thyroiditis by CD4+CD45RC
peripheral cells and CD4+CD8
thymocytes therefore does not support the view that the mechanism of regulation involves a
switch from a T helper cell type 1 (Th1) to a Th2-like response, but rather relies upon a specific suppression of the autoimmune responses involving TGF-
and IL-4. The observation that the same two cytokines were implicated in the protective mechanism, whether thymocytes or peripheral cells were used to prevent autoimmunity, strongly suggests that the regulatory cells from both sources act in the same way and that the thymocytes are programmed in
the periphery for their protective role. The implications of this result with respect to immunological homeostasis are discussed.
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Introduction |
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Rats and mice that are relatively T cell deficient for
genetic reasons, or that are made so experimentally,
develop a range of tissue-specific autoimmune diseases that
can be prevented by the transfer of the appropriate T cell
subset from syngeneic, nonlymphopenic donors. These
observations demonstrate that self-tolerance is an active
process and is not achieved entirely by a combination of
deletion of autoreactive T cells intrathymically and of
clonal anergy, deletion, or indifference in the periphery
(1). Studies in this laboratory have shown that insulin-
dependent diabetes is induced in a normal strain of laboratory rat by thymectomy at 6 wk of age followed by four
equal doses of 250-rad 137Cs -irradiation at 2-wk intervals.
Rats develop disease over the ensuing 10-12 wk, characterized by infiltration of pancreatic islets and selective destruction of the
cells in virtually all males and 70% of females. Disease is prevented in ~50% of rats reconstituted
shortly after the last irradiation with peripheral CD4+ cells
from syngeneic donors. Phenotypic analysis of these cells identified a subset of cells that protected 100% of recipients from diabetes as being CD4+CD45RC
TCR-
/
+RT6+
(6). Some of the cells with this phenotype have been
shown to produce IL-4 on activation in vitro (7) and provide B cells with help for secondary antibody responses (8),
raising the possibility that the prevention of diabetes involved a switch from a proinflammatory, cell-mediated,
Th1 response to a nonpathogenic humoral one. The peripheral T cells that prevented diabetes in these experiments have a memory phenotype, suggesting that they are
primed in the periphery to some undefined antigen. However, when CD4+CD8
thymocytes were assayed for their
ability to prevent diabetes in this system, it was found that
these were much more potent in regulating the disease than
were the peripheral memory cells (9). This unexpected
finding raised questions about the mechanisms of disease
prevention by the cells from the two sources and the developmental relationship between them. To examine these
questions, we have studied another lymphopenia-induced
autoimmune disease, which unlike the autoimmune diabetes has a strong humoral component.
In studies that originally described the development of
autoimmunity in normal rats after thymectomy and irradiation (TxX protocol), autoimmune thyroiditis, characterized by leukocytic infiltration of thyroid glands and the
development of high titers of antithyroglobulin (Tg)1 antibodies, was induced in PVG.RT1c strain rats (10, 11). In
these same studies it was shown that reconstitution of these
lymphopenic rats with splenocytes from normal syngeneic
recipients could prevent disease development. However,
the phenotype and function of the regulatory T cells that
prevent it has not been determined previously. In this study we show that, although thyroiditis in such rats is characterized by a humoral, anti-Tg response, it is the CD4+
CD45RC subset that is responsible for protection from
disease. As noted above, this same subset prevents the cell-mediated diabetes observed in TxX PVG.RT1u rats (6).
Further similarities in the mechanisms that prevent the two
diseases are shown by the observation that, as with the prevention of diabetes, CD4+CD8
thymocytes are also very
potent at preventing autoimmune thyroiditis. Finally, we
show that the cytokines IL-4 and TGF-
both play an essential role in the mechanism of protection by both peripheral CD4+CD45RC
T cells and CD4+CD8
thymocytes, strongly suggesting that the same cellular mechanism is involved.
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Materials and Methods |
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Monoclonal Antibodies.
The mouse mAbs used in these studies were as follows: W3/25 (anti-rat CD4; reference 12); OX6 (anti-rat class II MHC; reference 13); OX8 (anti-rat CD8; reference 14); OX12 (anti-ratAnimals and Induction of Thyroiditis.
3-12-wk-old female PVG.RT1c (PVG) rats were obtained from the specific pathogen-free breeding facilities of the Medical Research Council Cellular Immunology Unit (Oxford, UK). Thyroiditis was induced in female PVG rats as has been previously described (11). In brief, 3-wk-old female rats were thymectomized and given four 275-rad doses of 137CsIsolation of T Cell Subpopulations.
Rat thoracic duct lymphocytes (TDLs) were obtained by cannulation of the duct (22). Cells were collected at 4°C overnight into flasks containing PBS and 20 U/ml heparin. Rat lymphocyte populations were negatively selected from TDLs using a rosetting technique as described elsewhere (23) or using magnetic beads (Dynal). CD4+ T cells were isolated by depletion of B cells and CD8+ T cells using the mAbs OX12, OX8, and OX6. To obtain CD4+CD45RCDetection and Quantitation of Anti-Tg Antibodies in Sera of Rats.
Sera from TxX rats were assayed every 2 wk for anti-Tg antibodies between 4 and 12 wk after the last irradiation by specific ELISA. 96-well microtiter plates were coated overnight with purified rat Tg (20 µg/ml) and blocked for 30 min with 1% BSA in PBS. Sera of individual rats at four fivefold dilutions starting at 1:10 were incubated for 2 h at room temperature. Rat IgG was detected using anti-rat IgG alkaline phosphatase conjugate (Sigma Chemical Co.) for 1 h at room temperature, and different rat Ig isotypes were detected using biotinylated mAbs (1 µg/ml) specific for IgG1 (PharMingen), IgG2a (Serotec Ltd.), and IgG2b (PharMingen) for 1 h at room temperature followed by a 1 h incubation with avidin alkaline phosphatase (Sigma Chemical Co.). Plates were washed three times between each step with 0.05% Tween 20/PBS. The assay was developed using enzyme substrate 4-nitro-phenyl phosphate (5 mg/ml; Sigma Chemical Co.) for 15 min at room temperature before reading the OD at 405 nm. Anti-Tg antibody titers were quantified by comparison with a standard serum pooled from TxX rats with thyroiditis and high anti-Tg antibody titers and expressed as a percentage of this standard. The level of nonspecific binding found in normal PVG serum represented a titer of ~0.1% in the assay, and therefore only sera with titers >0.3% were considered to contain specific anti-Tg antibodies.Histological Analysis.
Whole thyroids attached to cricoid cartilage were dissected out and fresh frozen in O.C.T. embedding medium (Sakura Finetek U.S.A. Inc.). 10-µm sections were cut from frozen blocks and stained with hematoxylin and eosin.Statistical Analysis.
Incidence of thyroiditis development in two different groups of TxX PVG rats was compared for statistically significant differences using Fisher's exact test for consistency in a 2 × 2 table. ![]() |
Results |
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Previous studies have
shown that normal rats with no tendency to spontaneously
develop autoimmunity will develop organ-specific autoimmunity in a strain-dependent fashion if thymectomized and given four equal doses of -irradiation at 2-wk intervals.
After thymectomy and irradiation, female PVG rats spontaneously develop autoimmune thyroiditis characterized by
extensive infiltration of thyroid glands and development of
high titers of anti-Tg antibodies (10, 11). In contrast, similar treatment of PVG.RT1u strain rats, differing from PVG
rats only in their MHC haplotype, results in the development of a fatal insulin-dependent diabetes 4-10 wk after
the last irradiation at a high incidence in males (6). Studies
of the pathogenesis of diabetes in both TxX PVG.RT1u
rats and the nonobese diabetic (NOD) mouse strongly implicate a Th1 cell-mediated mechanism, since CD8+ T
cells are required for disease development (6, 24) and manipulations that antagonize development of Th1 responses
protect NOD mice from disease development (25).
To further characterize the pathogenesis of thyroiditis development in TxX PVG rats, the use of different IgG isotypes in the anti-Tg antibody response was analyzed. In the rat, antibodies of the IgG1 isotype, like those in the mouse, are associated with a Th2 response, whereas IgG2b isotype synthesis, in contrast to that in mice, is indicative of a Th1 response. The IgG2a isotype is not preferentially favored by either Th1 or Th2 reactions (28, 29). Although no anti-Tg IgG of any isotype was detectable in the sera of normal rats (data not shown), sera of TxX rats with thyroiditis consistently contained high titers of anti-Tg IgG of the IgG1 isotype, with little specific IgG2a or IgG2b antibodies (Fig. 1 A). This profile of anti-Tg IgG isotype usage contrasted significantly with that of normal PVG rats immunized with Tg in CFA, an adjuvant known to induce Th1 responses. Normal 12-wk-old PVG rats immunized with Tg (50 µg/rat) in CFA developed anti-Tg IgG of the IgG2b isotype, with little anti-Tg IgG1 (Fig. 1 A).
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Because the isotype of anti-Tg IgG antibodies in TxX rats indicated that the pathogenesis involved the activity of Th2 cells, the requirement for CD8+ T cells in the development of thyroiditis was tested. PVG rats were depleted of CD8+ cells by injection, at the time of thymectomy and 1 wk after thymectomy, with purified depleting anti-CD8 mAb OX8 in PBS (0.5 mg/injection). Control rats received injections of PBS alone. Although treated rats were still depleted of TCR+ CD8+ cells 12 wk after their last irradiation (data not shown), they developed thyroiditis at a similar frequency to control rats (Fig. 1 B) (P > 0.24). In principle, it is possible that residual CD8+ cells, whose presence was not readily detectable by FACS® analysis, mediated disease development. However, it is notable that similar depletion of CD8+ cells in PVG.RT1u rats was sufficient to completely prevent diabetes, suggesting that the depletion regime was effective (6).
Reconstitution of TxX PVG Rats with Syngeneic CD4+ CD45RCIn previous studies of autoimmunity in TxX rats, development of
diabetes could be prevented in ~50% of PVG.RT1u rats by
their reconstitution with unfractionated CD4+ T cells from
normal syngeneic donors (6). Similarly, thyroiditis development was prevented in TxX PVG rats by their reconstitution with unfractionated splenocytes (30). In the former
case, protection from diabetes development was shown to
be mediated by the CD4+CD45RC subset of CD4+ T
cells and antagonized by the CD4+CD45RC+ subset. This
antagonism explained why unfractionated CD4+ T cells
protected only some recipients while, in contrast, all prediabetic rats given the CD4+CD45RC
subset were free of
disease. Cells that share this protective CD4+CD45RC
phenotype provide B cells with help for secondary antibody responses (28) and produce IL-4 after activation in
vitro (7) and therefore have some of the characteristics of
Th2 cells. In principle then, protection from diabetes could
have reflected a switch from a cell-mediated to a humoral
response toward islet cell autoantigens. In contrast to the
cell-mediated mechanisms implicated in the pathogenesis of
diabetes, the IgG isotypes of anti-Tg responses in rats with
thyroiditis indicate the activity of Th2 cells and this observation calls into question the possible involvement of a Th1 to
Th2 switch in preventing these autoimmune diseases. However, the preceding data did not exclude the possibility that different subsets of CD4+ T cells were involved in the prevention of these two diseases. To examine this possibility, a
comparison was made of the abilities of CD4+CD45RC
and CD4+CD45RC+ cells to prevent thyroiditis.
Consistent with previous studies (30), reconstitution of
TxX PVG rats with 107 unfractionated CD4+ TDLs
shortly after their last irradiation prevented development of
thyroiditis in a high proportion of recipients (Fig. 2). Significantly, however, TxX PVG rats reconstituted with 107
CD4+CD45RC+ TDLs shortly after their final irradiation
developed thyroiditis at the same frequency as control rats
(Fig. 2). In contrast, TxX rats injected with 107 CD4+
CD45RC TDLs shortly after their final irradiation were
completely protected from development of both serological
(Fig. 2) and histological (Fig. 3) signs of disease.
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Previous studies in this
laboratory showed that diabetes could be prevented in a
large proportion of TxX PVG.RT1u rats after their reconstitution with CD4+CD8 thymocytes (6). Further experiments indicated that these thymocytes were effective at
preventing diabetes at a significantly lower dose than required for peripheral CD4+CD45RC
T cells (9). To determine whether CD4+CD8
thymocytes were equally
potent at preventing thyroiditis they were tested for their
ability to control onset of this disease at different cell doses.
PVG rats, thymectomized at 3 wk of age and given four
doses of 275 rad
-irradiation, were reconstituted shortly
after their last irradiation either with purified CD4+CD8
thymocytes at doses of 107, 5 × 106, 106, 5 × 105, or 105
per rat, or with CD4+CD45RC
cells purified from TDLs
of 12-wk-old normal donors at doses of 107, 5 × 106, or
106 per rat. In agreement with studies of diabetes in
PVG.RT1u rats, most but not all rats reconstituted with 107
CD4+CD8
thymocytes were protected from development of thyroiditis (Fig. 2) and, significantly, these thymocytes were found to be highly potent in controlling the
disease. An indistinguishable level of protection was observed in recipients of a range of CD4+CD8
thymocyte
doses, between 5 × 105 and 107 cells/rat. Only at doses below 5 × 105 CD4+CD8
thymocytes was protection lost
(Fig. 4). In contrast, although recipients of 107 CD4+
CD45RC
cells were protected from disease development,
a low incidence of disease was observed in recipients of half
that number of CD4+CD45RC
cells, and rats reconstituted with 106 CD4+CD45RC
cells were not protected
at all (Fig. 4).
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Results from this present and previous studies (6) have
established that the regulatory subset of peripheral T
cells that prevent autoimmunity in TxX rats are CD4+
CD45RCRT6+TCR-
/
+, and furthermore, that CD4+
CD8
thymocytes are also a potent source of cells with a
similar regulatory capacity. However, the mechanisms by
which these different populations prevent autoimmunity
and whether the regulatory cells in the thymus and those in
the periphery represent the same lineage of cells has not
been examined until now. By using mAbs that specifically block the biological activities of TGF-
and IL-4 it was
possible to determine the role of these cytokines in the
protection of rats from development of thyroiditis following their reconstitution with either CD4+CD45RC
cells
or CD4+CD8
thymocytes. Groups of PVG rats, thymectomized neonatally and 1 wk later started on a regime of
four 275-rad doses of 137Cs
-irradiation at 2-wk intervals,
were reconstituted shortly after their last irradiation with
either 5 × 106 CD4+CD45RC
cells purified from TDLs
of normal 12-wk-old PVG rats or with 106 CD4+CD8
thymocytes purified from thymus of normal 6-wk-old syngeneic donors. Control rats received no cells. Groups of
rats reconstituted with cells were then treated with either
OX81 (2 mg/injection) or 2G.7 (2 mg/injection) mAbs
that specifically block the biological activities of rat IL-4
(19) and rat TGF-
(31) respectively, while other groups
received isotype-matched negative control mAbs at the
same dose. Rats were injected with mAb 1 d before and 1 d
after reconstitution and then twice weekly thereafter for 4 wk.
This length of treatment was chosen because previous studies had shown that rats are protected from disease development when reconstituted with lymphocytes up to 14 d after the last irradiation. Development of thyroiditis in rats
reconstituted 4 wk after the last irradiation was unaffected
by the injection of the regulatory T cells (30), suggesting
that these cells have, at most, a 4-wk window after the final
irradiation in which they can affect disease development.
TxX rats reconstituted with a dose of 5 × 106 CD4+
CD45RC cells and either untreated or treated with isotype-matched negative control mAbs were effectively protected from development of thyroiditis, with only 7 out of
32 rats developing any disease compared with 25 out of 32 controls rats (Fig. 5 A). However, treatment of TxX rats
with mAbs specific for either TGF-
or IL-4 after their reconstitution with CD4+CD45RC
cells developed thyroiditis at a frequency comparable with that of control rats
(Fig. 5 A). 9 out of 13 TxX rats reconstituted with
CD4+CD45RC
cells and treated with anti-TGF-
mAb
developed anti-Tg autoantibody responses, whereas all rats
similarly reconstituted but treated with anti-IL-4 mAb
made anti-Tg IgG. Significantly, although 7 out of 30 rats
reconstituted with 106 CD4+CD8
thymocytes were protected from thyroiditis development (Fig. 5 B), rats similarly reconstituted but treated with anti-TGF-
or anti-IL-4
mAbs developed disease at a frequency similar to that of
controls. Of those TxX rats reconstituted with 106
CD4+CD8
thymocytes, 8 out of 10 anti-TGF-
mAb-treated rats and 12 out of 15 anti-IL-4 mAb-treated rats
developed thyroiditis (Fig. 5 B), compared with an incidence of 16 out of 22 control rats.
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IL-4 has long
been known to play a major role in developmental regulation of Th1 and Th2 immune responses, and in vivo blockade of its biological activity during immune responses is
therefore likely to affect the balance of Th1/Th2 T cell development. Therefore, it was possible that treatment of
TxX rats with cytokine-blocking mAbs was altering the
development of disease and consequent involvement of
Th1/Th2 T cell subsets to a response type that neither
CD4+CD45RC cells nor CD4+CD8
thymocytes could
regulate. To examine this possibility, the IgG isotype of
anti-Tg responses in reconstituted TxX rats treated with
anti-IL-4 mAb or anti-TGF-
mAb was assessed to determine whether there had been a change in the relative activity of Th2 and Th1 cells in these rats. As the data show,
the IgG isotypes in anti-Tg responses of rats reconstituted
with either CD4+CD45RC
cells or CD4+CD8
thymocytes and treated with either anti-IL-4 or anti-TGF-
mAbs were the same as those of controls (Fig. 6). Anti-Tg
responses of anti-cytokine-treated rats were predominantly
of the Th2-associated IgG1 isotype, with little or no IgG2b
anti-Tg detectable. Therefore, these data support the view
that blockade of either IL-4 or TGF-
with neutralizing
mAbs in TxX rats reconstituted with CD4+CD45RC
T
cells or those given CD4+CD8
thymocytes abrogates the
protection mediated by these populations with no modulation of the disease process itself.
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Discussion |
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There are close similarities between the autoimmune thyroiditis reported here and the insulin-dependent diabetes
described in earlier papers from this laboratory. In both cases
a tissue-specific autoimmune disease develops after experimentally induced lymphopenia, and the disease can be prevented by the injection of peripheral CD4+CD45RC T
cells or CD4+CD8
thymocytes from normal syngeneic
donors (reference 9 and Fig. 2). However, there are significant differences that are informative in determining the
mode of action of the regulatory T cells that prevent these diseases.
The diabetes that develops in lymphopenic PVG.RT1u
rats has the features of a cell-mediated disease in which
CD8+ T cells play an essential role. In contrast, the autoimmune thyroiditis in TxX PVG rats, described in this
paper, is characterized by a strong humoral component.
CD8+ T cells are not required for the development of this
disease (Fig. 1 B) and the anti-Tg IgG antibodies that develop are predominantly of the IgG1 isotype (Fig. 1 A). In
principle, this observation could be explained if PVG rats
are able to make only a Th2-type immune response to Tg.
However, active immunization of PVG rats with Tg/CFA
induces anti-Tg antibodies that are mainly IgG2b rather than IgG1 (Fig. 1 A) demonstrating that PVG rats, when
appropriately challenged, are able to make an anti-Tg antibody response characteristic of Th1 activity. Despite the
fact that the diabetes and thyroiditis that develop in TxX
rats have characteristics of Th1 and Th2 autoimmune responses respectively, the same CD4+CD45RC subset of
peripheral T cells was able to completely prevent both diseases (Fig. 2 and reference 6). This observation counters the suggestion that regulatory T cells prevent diabetes in
TxX PVG.RT1u rats by inducing a Th1 to Th2 switch in
the autoimmune response. Similarly, the suppression of autoantibody synthesis in the TxX PVG rats by the transfer of
CD4+CD45RC
T cells is incompatible with such a
change in T cell function since the anti-Tg autoantibody in
unprotected rats already has the characteristics of a Th2 response. Taken together, the data from the two autoimmune diseases do not support the proposal that the control
of autoimmunity involves a change in the balance between Th1 and Th2 responses to an autoantigen (32, 33). Instead, the regulatory T cells that prevent organ-specific autoimmune diseases appear to suppress autoimmune T cells completely. A recent publication draws the same conclusion
(34).
The experiments on the relative potency of peripheral
CD4+CD45RC T cells and CD4+CD8
thymocytes in
controlling thyroiditis in TxX PVG rats indicated that the
latter cells were effective at one-tenth the dose of the
peripheral ones (Fig. 4), a result that closely resembles the findings on the control of autoimmune diabetes in
PVG.RT1u rats (9). The experiments on the mode of action of these regulatory cells in preventing thyroiditis indicated that both IL-4 and TGF-
are involved in this mechanism (Figs. 5 and 6), and this was true whether peripheral
T cells or thymocytes were used to control the disease.
This result suggests that the differences in the numbers of
protective cells from these two sources, required to control
autoimmunity in TxX rats, cannot be explained in terms of
differences in the way in which they mediated protection.
This conclusion is relevant when considering the nature of
the homeostatic mechanism that controls the size of the
regulatory T cell population. When CD4+CD8
thymocytes were used to prevent these diseases, they were
injected into recipients that were highly deficient in regulatory T cells. Consequently, a homeostatic control mechanism would be expected to permit a relatively large number of thymocyte precursors to differentiate into cells with
regulatory function. In contrast, there is evidence (see below) that the mature peripheral CD4+CD45RC
regulatory T cells, which were obtained from normal, healthy donors, represent a population whose frequency has been
subject to homeostatic control within these nonlymphopenic animals. It appears that few extra regulatory T cells
(T reg) can be recruited from the peripheral T cell pool
when donor CD4+ T cells are transferred into TxX recipients. Consequently, peripheral T cells are a less potent
source of T reg than are thymocytes that have not been exposed to the homeostatic mechanism that determines the
size of the peripheral T reg population.
The intrinsic nature of the homeostatic mechanism is not
directly addressed by these experiments but the data are
best explained by a process in which the number of thymocytes that develop into peripheral regulatory T cells is
determined by the requirement that this number should be
just adequate to control the potentially autoreactive T cells
that are part of the normal T cell repertoire (6). As with the
previous observations on the control of diabetes in TxX
rats (9), the data in Fig. 4 show that the percentage of rats
protected from autoimmunity by CD4+CD8 thymocytes
is very insensitive to the dose of thymocytes used and that
the majority of, but not all, rats are protected from thyroiditis by unfractionated CD4+ peripheral T cells. In the
experiments on diabetes in TxX rats, we proposed that
these findings indicated that there is a delicate balance between the number of autoreactive T cells in the periphery and the number of T reg that control them, with the latter
population being in a slight functional excess. This interpretation was supported by the close agreement between
the experimental findings and a mathematical model based
on this concept (9). The data presented here on autoimmune thyroiditis are also compatible with this interpretation. The most economical explanation of how this balance
between the two populations of T cells is achieved is that
exposure of regulatory T cell precursors and potentially autoreactive T cells to the specific autoantigen leads to an interaction between them and that this interaction ceases
only after the regulatory T cells so generated are sufficient
to control the autoreactive T cells. This hypothesis would
require the presence of the relevant autoantigen in the periphery to transiently stimulate the autoreactive T cells. Consistent with this proposed mechanism, we have recently shown that peripheral regulatory T cells that prevent
thyroiditis are not found in rats whose thyroids have been
ablated by 131I treatment in utero (Seddon, B., and D. Mason, manuscript submitted for publication). In summary,
the data indicate that the mature CD4+CD8
thymocyte
population contains a high frequency of cells that have the
potential to differentiate in the periphery into regulatory T
cells that prevent organ-specific autoimmunity. In normal
animals this differentiation is homeostatically controlled,
such that the number of regulatory T cells generated is just
sufficient to prevent disease. The generation of regulatory
T cells in the periphery from thymic migrants does not take
place in the absence of the target autoantigen, so the numerical balance between autoreactive and regulatory T
cells is not established. Under such circumstances, the engraftment of the relevant organ leads to its rejection (35).
The immunosuppressive effect of TGF- has been reported in a number of systems (36) and has been implicated in suppression of both cell-mediated responses, as in
models of inflammatory bowel disease (41) and humoral
autoimmunity induced by mercuric chloride (31). Consequently, the essential role that it plays in the prevention of
autoimmune thyroiditis in TxX rats is not unexpected. As
TGF-
prevents T cell activation but does not kill activated cells, it appears that T reg suppress but do not eliminate autoreactive T cells. This conclusion is compatible
with the observation that such autoreactive cells form part
of the normal T cell repertoire, but it raises the question of
the value of a mechanism that preserves self-tolerance but
also preserves the autoreactive cells. As discussed elsewhere
(42), the establishment of self-tolerance by a mechanism that irreversibly silences all potentially autoreactive T cells would virtually completely deplete the T cell repertoire.
The implication seems to be that the maintenance of self-tolerance by a dominant regulatory process is required to
preserve a T cell repertoire that can react to essentially all
foreign MHC-binding peptides. If this is so, then T reg
would be required to inhibit autoreactive T cells when
these are stimulated by self-antigens but not by foreign
ones. This distinction may come about because peripheral
autoreactive T cells for self-antigens have low affinity receptors for self-epitopes since T cells with high affinity receptors have been intrathymically clonally deleted. It is notable in this context that there is compelling evidence for
the intrathymic expression of many "tissue-specific" autoantigens, including insulin and Tg (43) and that polymorphisms in the level of expression influence susceptibility (46).
A deficiency of IL-4 has been implicated in the pathogenesis of insulin-dependent diabetes mellitus in the NOD
mouse (33, 47, 48) but this view is not universally accepted
(49). Furthermore, IL-4 plays no essential part in the prevention of inflammatory bowel disease in mice (41), and
regulatory T cells that prevent this condition appear not to
produce this cytokine in vitro (50). In contrast, in our current experiments the neutralization of IL-4 by specific antibody completely abrogated the protection from autoimmune thyroiditis provided by the transfer of syngeneic CD4+ T cells. Although this result would at first appear to
contradict the view that autoimmune thyroiditis is mediated by a humoral Th2 mechanism, it is possible that the
requirement for IL-4 by regulatory and autoreactive T cells
may occur at different times, even if the same anatomical
site is implicated. Support for this comes from the observation that suppression of disease by reconstitution of rats
with T cells is only successful up to 2 but not 4 wk after the
last irradiation (30), whereas development of anti-Tg antibodies in TxX PVG rats occurs only between 4 and 12 wk
after the last irradiation. Despite this finding, we cannot
conclude with certainty that there was a deficiency of IL-4
in the rats that developed thyroiditis, since a deficiency in
TGF- would have sufficed to cause the disease. The IgG1
isotype of the anti-Tg antibody in the rats with thyroiditis
is compatible with the presence of IL-4, but this antibody
isotype is not strictly IL-4-dependent (51). Despite these
uncertainties, the experimental data in Figs. 5 and 6 indicate that an anti-IL-4 antibody-induced IL-4 deficiency does result in the development of autoimmune thyroiditis
in TxX rats.
Relevant to discussions of the possible functional role of
IL-4 in the prevention of autoimmunity, it is significant
that mouse T cells activated in the presence of IL-4 secrete
high levels of TGF- during both primary and secondary
stimulations (52). This raises the possibility that IL-4 is a
growth factor for regulatory T cells, whereas TGF-
acts as
the effector cytokine in the suppression of autoimmune responses such that a deficiency of either cytokine will result
in breakdown of regulation. Consequently, these data are
in accord with those studies that implicate a deficiency of IL-4 in the pathogenesis of insulin-dependent diabetes
mellitus in NOD mice and suggest that it plays an important role in the prevention of organ-specific autoimmune
diseases. The failure to show a similar involvement of this
cytokine in the prevention of inflammatory bowel disease
indicates that regulatory T cells do not necessarily form a
functionally homogeneous population.
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Footnotes |
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Address correspondence to Benedict P. Seddon, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK. Phone: 44-181-959-3666; Fax: 44-181-913-8531.
Received for publication 27 July 1998 and in revised form 9 November 1998.
Thanks go to Liz Darley, Ruth Goddard, Michael Puklavec, and Steve Simmonds for technical assistance. Thanks also to Catarina Amorim, Francisco Ramirez and Leigh Stevens together with other members of the Cellular Immunology Unit for discussion and interest. ![]() |
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