By
From the * Laboratory of Immunology, Division of Hematologic Products, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20852; and Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892-1152
While it is generally believed that the avidity of the T cell antigen receptor (TCR) for self antigen/major histocompatibility complex (MHC) determines a thymocyte's fate, how the cell
discriminates between a stimulus that causes positive selection (survival) and one that causes
negative selection (death) is unknown. We have previously demonstrated that glucocorticoids
are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking. To examine the role of glucocorticoids during MHC-dependent selection, we examined thymocyte development in organ cultures in which corticosteroid biosynthesis was inhibited. Inhibition of glucocorticoid production in thymi from /
-TCR transgenic mice resulted in the
antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC
with sufficient avidity to result in positive selection. Furthermore, inhibition of glucocorticoid
production caused an increase in apoptosis only in CD+CD8+ thymocytes bearing transgenic
TCRs that recognized self antigen/MHC. These results indicate that the balance of TCR and
glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.
Rigorous selection processes in the thymus prevent
nonfunctional or harmful T cells from reaching the
periphery. Thymocytes that express a TCR with high
avidity for self peptides presented by self MHC-encoded
molecules are eliminated by TCR-mediated apoptosis (negative selection). Thymocytes bearing TCRs with lowto-moderate avidity for self peptide/MHC are rescued
(positive selection) from the default apoptosis pathway undergone by thymocytes that have not successfully rearranged their TCR genes or that express a receptor with
subthreshold avidity for self-MHC (1, 2). How ligandinduced signaling through the TCR can lead to both rescue from death, in the case of positive selection, and death,
in the case of negative selection, is unclear.
Occupancy of the glucocorticoid receptor (GR) is a potent means of inducing apoptosis in thymocytes (3).
However, stimulation with glucocorticoids does not necessarily always result in thymocyte death. Paradoxically, lowto-moderate concentrations of glucocorticoids antagonize
TCR-mediated apoptosis of T cell hybridomas and thymocytes (6). Furthermore, transgenic mice generated to
express antisense transcripts to the 3 In this report, we address this prediction by analyzing the
effect of preventing thymus glucocorticoid biosynthesis on
the survival of thymocytes bearing transgenic TCRs of
known antigenic specificity. The results indictate that endogenous glucocorticoids prevent thymocyte apoptosis only
when the TCR is capable of recognizing self antigen/
MHC with sufficient avidity to normally result in positive
selection.
Mice and Reagents.
RAG-2 Thymic Organ Culture.
Thymic organ cultures were performed
as described, in serum-free Nutridoma-SP medium (Boehringer
Mannheim Corp., Indianapolis, IN) supplemented with 20 mM
Hepes, 100 mM nonessential amino acids, 1 mM sodium pyruvate, and 50 mM 2-mercaptoethanol (organ culture medium)
(11). Thymi from 1-2-d-old mice were separated into lobes and
cultured with 200 µg/ml of freshly diluted metyrapone, or ethanol control on a Millipore filter floating on a gelfoam sponge in
organ culture medium. The sex of each neonate was determined microscopically before removal of the thymus. In some experiments, 10 TUNEL Assay.
A modified form of the TUNEL assay (14)
was used to detect fragmented DNA in apoptotic thymocytes (9).
In brief, thymocytes were formaldehyde-fixed, permeablilized with
0.1% Triton X-100 and 0.1% sodium citrate, and subjected to a
modified in situ nick translation using fluorescein-dUTP (Boehringer Mannheim Corp.). Labeled cells were visualized by flow
cytometry with a FACScan®. To determine the amount of apoptosis in the DP population, thymocytes were gated for expression
of both CD4 and CD8 and analyzed for dUTP-FITC incorporation. Percent specific apoptosis was calculated by the formula:
untranslated region of
the GR in immature thymocytes revealed that glucocorticoids are critical during at least two points during thymocyte development: progression from the CD4
CD8
to
CD4+CD8+ stage, and maintenance of viability at the
CD4+CD8+ stage (9). In addition, we have previously
shown that thymic epithelial cells produce steroids, and
that addition of low concentrations of metyrapone, a selective inhibitor of corticosteroid synthesis (10), to fetal thymic organ culture enhances TCR-mediated deletion, an
effect that was reversed by the addition of corticosterone to
the cultures (11). Based upon these results, we have proposed that glucocorticoids participate in the antigen-specific development of thymocytes by preventing the TCR-mediated
deletion of cells bearing receptors with low-to-moderate
avidity for self antigen/MHC. A prediction that follows
from this hypothesis is that interfering with glucocorticoid
signaling should convert instances of positive selection
(TCR interaction with low-to-moderate ligands) to negative selection (deletion by apoptosis) because of the failure of glucocorticoids to antagonize the TCR-mediated signals.
/
H-2d and H-2b mice bearing
TCRs specific for H-Y/Db (12) were bred in our facilities by
crossing RAG-2
/
mice (H-2b, 129 background, (13)) with
H-Y/Db-specific TCR transgenic H-2d mice. TCR transgeneexpressing offspring were bred to RAG-2
/
mice to generate
offspring that were RAG-2
/
H-2bxd and expressed the transgenic TCR. These mice were interbred to generate timed pregnants or to establish lines of RAG-2-/- TCR transgenic mice
that were homozygous for either H-2d or H-2b. Metyrapone was
purchased from ICN Biochemicals (Cosa Mesa, CA). Antibodies
used for flow cytometry, anti-CD4, anti-CD8, anti-TCR, and
anti-MHC, were purchased from PharMingen (San Diego, CA).
9M corticosterone, diluted in ethanol, was added to
the cultures. Cultures were carried out in 3 ml of medium in 6-well
plates. Lobes were harvested after 1 or 3 d of culture and single
cell suspensions were prepared for counting and analyzed for
CD4, CD8, TCR, and MHC expression, or for TUNEL positivity, by flow cytometry with a FACScan® (Becton Dickinson,
Mountain View, CA).
The mutual antagonism model of thymocyte selection (6) postulates that in the absence of glucocorticoids,
occupancy of TCRs on CD4+CD8+ cells by ligands with
either low-to-moderate or high avidity leads to apoptosis
(negative selection). In the presence of glucocorticoids, however, thymocytes that recognize ligands with only low-
to-moderate avidity would survive and differentiate into
CD4+CD8 and CD4
CD8+ thymocytes (positive selection). Therefore, eliminating glucocorticoids during thymocyte development should result in the death of thymocytes that recognize self antigen/MHC with avidity
(low-to-moderate) that would otherwise lead to positive
selection. To test this, organ cultures were performed with
thymi from mice whose cells bear a transgenic
/
-TCR
specific for the male H-Y antigen in the context of the H-2b
MHC class I molecule (12). Thymocytes from H-2b male
mice bearing the H-Y/Db-specific receptor are normally
deleted, resulting in a dramatic reduction in the number of
CD4+CD8+ (double positive, or DP) cells (12). Thymocytes
from female mice are not deleted because they do not express the male H-Y antigen. However, in animals of the
H-2b, but not the H-2d, haplotype, DP thymocytes from
female mice undergo positive selection and mature into
clonotype-bearing CD4
CD8+ cells, indicating that this
transgenic TCR recognizes some as yet uncharacterized
peptide antigen plus an H-2b-encoded molecule with lowto-moderate avidity (15). Because of allelic exclusion, all
TCR-
chains expressed in these transgenic animals are
encoded by the
transgene, but incomplete allelic exclusion of the
chain results in a significant number of thymocytes that express endogenous rearranged TCR-
as
well as transgenic
(15). To eliminate this complication
and ensure that all of the thymocytes in these animals express only the transgenic TCR, RAG-2
/
mice, which
lack the rag-2 gene and therefore produce thymocytes that
do not rearrange endogenous TCR-
and -
genes (13),
were bred to express the transgenic H-Y/Db-specific TCR.
To address what effect a decrease in corticosteroid production would have on the antigen-specific fate of DP thymocytes, RAG-2/
H-2bxd TCR females and males
were bred and thymi from 1-2-d-old offspring were used
in organ culture experiments. Thymocyte development is
delayed in SCID mice and RAG-2
/
mice that express
a transgenic TCR; thymi of these newborn mice have the
developmental phenotype of thymi from fetal d17-18 of
normal mice (16 and our unpublished observation). We
have previously shown that glucocorticoids play a role in
the DN to DP transition (9). In newborn RAG-2
/
TCR transgenic mice, however, the large majority of thymocytes have already reached the DP stage, allowing us to
address the effects of metyrapone on antigen-specific selection. The thymi were separated into three pieces and cultured in the absence or presence of metyrapone. Metyrapone
is a selective inhibitor of the enzyme P450c11 that blocks
the conversion of biologically inactive deoxycorticosteroids
to the active forms, corticosterone or cortisol, and has been
used clinically to treat hypercortisolism and as a test for the
pituitary's response to decreased levels of plasma cortisol
(10). Thymic organs cultures were performed in serumfree medium to avoid the addition of any steroids present in fetal calf serum or any steroid-binding proteins. The addition of metyrapone caused a dramatic decrease in thymocyte recovery (<20% of the medium control) in H-2b
thymi from female animals (Fig. 1). This was largely due to
the loss of DP cells, as the number of CD4
CD8
(double
negative, DN) cells was the same in the medium and
metyrapone-treated groups (Fig. 2). As in previous studies
(9, 11), the effect of metyrapone was significantly reversed
by the addition of a physiologic concentration (10
9 M) of
free corticosterone. To determine if occupancy of the
transgenic TCR by ligand was required for the decrease in
cell recovery, thymi from H-2d littermates, which do not
express a ligand recognized by the transgenic TCR (15),
were evaluated. In contrast to the H-2b thymi, metyrapone
had little if any effect when added to organ culture of
thymi with the nonselecting haplotype (Figs. 1 and 2), or
to RAG-2
/
nontransgenic thymi, which consist of
only CD4
CD8
cells (13). Notably, the addition of
metyrapone to female H-2b RAG-2
/
transgenic thymi
lowered cell recovery to levels almost equal to that from
RAG-2
/
nontransgenic thymi cultured in medium alone
(Fig. 1). Similar results were obtained in experiments performed with thymi from female SCID mice expressing the
transgenic H-Y/Db-specific TCR (data not shown). Together, these results demonstrate that inhibition of endogenous corticosterone production results in a marked loss of
thymocytes that have TCRs with functionally significant
(as judged by their ability to mediate positive selection) avidity for self but not those that do not.
Inhibition of Thymic Corticosteroid Synthesis Results in Apoptosis of Only Thymocytes That Bear TCRs That Recognize Self Antigen/MHC.
Antigen-specific thymocyte deletion is
due to the induction of apoptotic cell death (17, 18).
Therefore, experiments were performed to determine if the
decrease in thymocyte number when corticosteroid production was inhibited was due to apoptosis of DP thymocytes. Cells undergoing apoptotic death acquire single- and double-strand DNA breaks, which can be detected by
techniques in which labeled oligonucleotides are incorporated into the damaged DNA. We have used a modified
version of the original TUNEL assay in which harvested
thymocytes are stained with antibodies to cell surface molecules such as CD4 and CD8, permeabilized, and incubated with DNA polymerase I, allowing the incorporation of
fluoresceinated dUTP into nicked DNA. The cells are
analyzed by flow cytometry and the number of TUNELpositive cells quantitated. A representative example of profiles of thymocytes from a RAG-2/
H-2b or a RAG2
/
H-2d thymus cultured in the absence or presence of
metyrapone for 24 h is shown in Fig. 3. In the nonselecting
H-2d haplotype, the background level of spontaneous cell
death was unaffected by the addition of metyrapone. In
contrast, metyrapone caused a substantial increase in DP
TUNEL-positive cells compared to medium alone in DP
thymocytes of the H-2b haplotype. When the data from
multiple experiments were averaged (Fig. 4), it was found
that there was little if any increase in spontaneous apoptosis
in DP cells when thymi from RAG-2
/
H-2d mice expressing the H-Y/H-Db-specific TCR were cultured with
the corticosteroid-synthesis inhibitor. In contrast, in thymi
of H-2b littermates there was an induction of almost 20%
specific apoptosis. Therefore, inhibition of local corticosteroid production in the thymus specifically causes the
apoptotic death and deletion of DP cells only when their
TCRs recognize self.
Positive selection of thymocytes is an essential step in the formation of the T cell antigen-specific repertoire. It is generally believed that it is the avidity of TCRs for self antigen/MHC that determines the fate of immature (1, 2, 19, 20). Low to moderate avidity interactions result in thymocyte survival and differentiation (positive selection), while high avidity interactions result in apoptosis (negative selection). Given that there will be a continuum of TCR avidities for self, some mechanism must be present to set the threshold that distinguishes positive from negative selection. How the cell interprets the avidity of the TCR- ligand interaction is unknown. One possibility is that there are qualitative differences in the signals generated in response to TCR-ligand interactions of different avidities (21). The different signals generated would result in either rescue from, or induction of, apoptosis. Another possibility is that varying the avidity of the TCR-ligand interaction results in a quantitatively, but not qualitatively, variable signal, which induces thymocytes to both differentiate and undergo apoptosis. A third, not mutually exclusive, possibility is that another receptor-mediated stimulus, such as that mediated by the glucocorticoid receptor, may prevent apoptosis and allow positive selection to occur. The observation that glucocorticoids can antagonize TCRmediated apoptosis in T cell hybridomas and thymocytes has provided the basis for a mutual antagonism model of thymocyte development (6). This model proposes that the quantitative balance between TCR and GR signaling sets the threshold of TCR avidity for self that distinguishes positive from negative selection. Whereas low-to-moderate avidity TCR interactions are antagonized by glucocorticoids, allowing positive selection to proceed, high-avidity TCR interactions produce a signal too potent to be antagonized. Death by neglect may result from GR stimulation in the absence of TCR-mediated signaling.
The results in this report support the mutual antagonism
model by demonstrating that TCR avidity for self antigen/
MHC is in fact not the only factor that determines the fate
of immature thymocytes: inhibition of glucocorticoid production caused the apoptotic death of thymocytes bearing a
transgenic /
-TCR that would normally have been positively selected, but had little effect in mice that lacked expression of the appropriate MHC. Although the mechanism for the antagonism of TCR-mediated thymocyte death by glucocorticoids is not yet known, there are several clues
that can be taken from previous work with T cell hybridomas and mature peripheral T cells. Glucocorticoids inhibit
activation-induced apoptosis in these cells by inhibiting the
upregulation of Fas ligand (24, 25). While Fas and Fas
ligand do not appear to have a similar role in the TCRmediated death of thymocytes (26), a study with mice in
which CD30, a member of the gene superfamily that includes Fas, was deleted by homologous recombination has suggested that this molecule may be required for antigenspecific thymocyte deletion (27). Among the many possibilities, corticosteroids may regulate, for example, the expression of CD30 or its ligand.
The data presented here demonstrate that locally-produced corticosteroids are important for preventing TCR occupancy by low-to-moderate avidity ligands from causing apoptotic death (negative selection). Since the rescued cells go on to be positively selected (15), thymic-derived corticosteroids are likely to influence the peripheral antigen-specific T cell repertoire. This may be directly testable by analyzing the immune response of mice in which the thymocyte response to glucocorticoids is blunted because they express antisense GR driven by the proximal lck promoter (9). Such studies are currently being pursued in our laboratory.
Address correspondence to Melanie S. Vacchio, Food and Drug Administration, Center for Biologics Evaluation and Review, Suite 200N/HFM 538, 1401 Rockville Pike, Rockville, MD 20852-1448.
Received for publication 14 March 1997 and in revised form 11 April 1997.
We thank Dr. E. Shores for providing the TCR transgenic mice, Dr. J. Zuniga-Pflucker for providing the
RAG-2/
mice, and Drs. A. Singer, R. Hodes, R. Germain, L. King, A. Weissman, A. Rosenberg, and
E. Shores for critical review of this manuscript.
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