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*
Pediatric Immunology, The Children’s University Hospital, Basel University, Basel, Switzerland;
Department of Research, Kantonsspital Basel, Basel University, Basel, Switzerland; and
The Basel Institute for Immunology, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßTCR lineage. Lymphoid cells differentiate in direct physical
contact with thymic stromal cells, and this interaction is not only
critical for thymocyte selection and maturation but is equally
essential for the induction of a functionally competent thymic stromal
compartment. The thymic primordium of mice arises bilaterally at day 10
of gestation (E10)4 by
fusion of the third pouch endoderm with ectoderm from the corresponding
branchial clefts. The two tissues are separated by a thin layer of
mesenchyme originating from the cephalic neural crest (1).
While the relative contribution of each germ layer to the mature thymic
epithelial compartment remains to be determined, a small subpopulation
of epithelial cells has been characterized that represents a common
precursor cell for mature cortical and medullary epithelium (2, 3). At day E11.5, hemopoietic precursor cells seed the thymus
anlage although its epithelial cells are not yet competent to fully
support T cell development (4). This capacity is only
achieved after further maturation when epithelial cells have
differentiated into distinct stromal cell types (reviewed in Ref.
5). Finally, the thymic microenvironment is composed of an
integrated network of epithelial reticular cells and nonepithelial
stromal cells (i.e., fibroblasts, macrophages, and interdigitating
reticular cells) each characterized by typical structural, antigenic,
and functional features. Notably, the organization of the thymic
epithelium differs from most other epithelial organs in the body:
rather than forming a sheet of cells positioned on a basement membrane,
thymic epithelial cells (TEC) form a three-dimensional meshwork
(6).
The space between the diverse thymic stromal cells is occupied by
thymocytes at different stages of development as defined by their
expression of CD4 and CD8 (7). The most immature
population of intrathymic T cell precursors lack CD4, CD8, and CD3
expression and are referred to as triple negative (TN). These cells can
be further classified into four independent subpopulations according to
their sequential expression of CD44 and CD25: early thymic immigrants
are CD44+CD25- (TN I) and
develop via a CD44+CD25+
(TN II) and a CD44-CD25+
(TN III) stage to immature thymocytes with a
CD44-CD25- (TN IV)
phenotype (8). Subsequently, thymocytes begin to express
transiently CD8 before become
CD4+CD8+ (double positive,
DP) cells, a population that constitutes the majority of thymocytes. At
this stage, DP cells express an ßTCR that renders them subject to
either positive or negative selection dependent on their Ag
specificity. Only a minority (
3%) of the DP cells are positively
selected to generate mature single positive (SP) helper
(CD4+CD8-) and cytotoxic
(CD4-CD8+) T cells
(9). The developmental progression from immature to mature
thymocytes occurs sequentially and in distinct thymic microenvironments
(10).
The typical architecture of the complex thymic stroma is critically dependent on intercellular communications. Early in ontogeny, fibroblastoid cells control the differentiation of mesenchymal cells to form a regular epithelial compartment. Depending on cell-cell interactions between developing thymocytes and stromal cells (termed thymic cross-talk), distinct microenvironments are created that allow all steps in T cell maturation to occur. Importantly, the lack of inductive signals from developing T cells prevents the formation of the distinct cortical and medullary microenvironments (11, 12). Consequently, the absence of a normal stromal compartment hinders the orderly maturation of thymocytes and impedes the establishment of self-tolerance (13, 14, 15). Conversely, diverse experimental models of autoimmunity, such as Chagas disease, scleroderma, lupus erythematosus, and insulin-dependent diabetes, have been correlated with an obvious disorganization of thymic stromal architecture (16).
Clonal deletion and the induction of anergy represent two major mechanisms that establish self-tolerance among thymocytes. Immunity to self-Ags is also prevented by the presence of thymus-derived T cells, which play an active role in regulating the autoreactive potential of cells that have neither been clonally deleted nor rendered anergic (17, 18, 19, 20). However, the relative importance of these mechanisms for the establishment of self-tolerance remains to be determined for particular Ags.
The cellular and molecular mechanisms responsible for many forms of autoimmune diseases have yet to be defined due to the fact that these conditions are polygenic and have different environmental triggers responsible for their clinical emergence. In contrast, monogenic autoimmune syndromes provide appropriate models to gain further in-depth insights into the complex molecular processes associated with the loss of self-tolerance. Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, also known as autoimmune polyglandular syndrome 1; OMIM 240300) is a rare autosomal recessive disease with no known HLA association. Along with the autoimmune lymphoproliferative syndromes 1 and 2 (21, 22), APECED is recognized as one of three autoimmune disorders known to be caused by a single gene defect.
The gene responsible for APECED has been designated AIRE (autoimmune regulator) and has been mapped to chromosome 21q22.3 (23, 24, 25). Mutations in the coding region of AIRE result in truncated proteins devoid of a normal function. The murine Aire gene has recently been cloned and mapped to chromosome 10, revealing a structural organization and sequence homology highly conserved to its human ortholog (26, 27, 28). Aire encodes a predicted protein of 552 aa that contains structural features that anticipate a role in gene transcription. The protein embodies a proline-rich region, four LXXLL motifs, two plant PHD zinc-finger domains (which are restricted to nuclear proteins including transcriptional coactivators and chromatin-modulating proteins of the polycomb and trithorax groups), and a SAND domain (a sequence present in Sp100, Aire, NucP41/75 and DEAF-1/suppressin) (29, 30, 31). Moreover, the AIRE gene product is localized to distinct spherical nuclear structures, further corroborating its putative function as a regulator of gene transcription (32).
The autoimmune manifestations of APECED encompass hypoparathyroidism, primary adrenocortical failure, and chronic mucocutaneous candidiasis (29). APECED is characterized by lymphocytic infiltrations, the presence of a wide variety of tissue-specific, T cell-dependent autoantibodies, and a yet-unidentified defect in T cell function (29, 33, 34).
To detail its role in thymic function and in T cell tolerance induction, we investigated the temporal and spatial expression of Aire in embryonic and adult mice and in models of positive and negative thymic selection. Here we report that Aire expression is restricted to a distinct subpopulation of TEC, and its expression requires thymocyte stromal cell-cell interactions, a critical prerequisite for the generation of an appropriate thymic architecture. Moreover, Aire expression is dependent on RelB function, and the number of Aire-positive thymic stromal cells correlates with the presence of thymocytes undergoing negative selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BALB/c, C57BL/6, Rag-2-/-, Tg
26,
B6.RAG-2-/- I-Ab, and
B6.RAG-2-/- I-Abm12 mouse
strains were housed at the Animal Facility of the Kantonsspital Basel
and the Basel Institute for Immunology, respectively, according to the
Institutional Review Boards. The B6.RAG-2-/-
I-Ab and B6.RAG-2-/-
I-Abm12 mice express the 3BBM74 TCR as a
transgene on the RAG-2null background (see Ref.
35).
Tissues
Thymic tissue was obtained from adult mice and from embryos after timed pregnancies where detection of the vaginal plug was considered as day 0 of gestation (E0). Thymic tissue from RelB-/- mice was kindly provided by Dr. Li Wu (Melbourne, Victoria, Australia) and Dr. Philippe Naquet (Marseille, France). Thymic tissue was embedded in OCT (Tissue-Tec, Miles, Elkhart, IN) for analysis by immunohistology and in situ hybridization (ISH).
Cell sorting
Thymocytes were obtained by tissue disruption using frosted glass slides, whereas TEC were prepared by enzymatic digestion of whole thymi as described elsewhere (36, 37). After enrichment of low-density cells by Percoll gradient for the sorting of TEC, cell subsets were stained with a combination of CD3, CD4, CD8, CD11c, and CD45 (all from PharMingen, San Diego, CA), as well as mAbs 29 (36), G8.8 (38), and CDR1 (39), respectively. The different lymphocyte and stromal subpopulations were sorted by flow cytometry (FACStar; Becton Dickinson, Mountain View, CA) to a purity of at least 98%.
RNA isolation and RT-PCR analysis
Total RNA was isolated from tissues or sorted cells using TRI
reagent (Molecular Research Center, Cincinnati, OH). Sorted cells were
supplemented with 10 µg of yeast transfer RNA (Life Technologies,
Basel, Switzerland) as a carrier. For the generation of cDNA, RNA was
treated with RNase-free DNaseI (Roche Molecular Biochemicals,
Gipf-Oberfrick, Switzerland) and then reverse transcribed using
SuperScript II reverse transcriptase with either oligo(dT) (for
tissues) or random hexamers (for sorted cells) as primers (Life
Technologies). For PCR, various amounts of cDNA were used with 1x
Taq PCR buffer (1.5 mM Mg2+), 0.5 U
Taq, 0.2 mM dNTP (all from Sigma, St. Louis, MO), and 0.4
µM of each oligonucleotide. The following oligonucleotide pairs were
designed from publicly available data: Aire, 5'-TGC ATA GCA
TCC TGG ACG GCT TCC and 5'-CCT GGG CTG GAG ACG CTC TTT GAG; as well as
5'-TCT ACT GAG TGC TGG GAA TGA G, and 5'-CAG GAA GAG AAG GGT GGT GTC
(see Fig. 3
B); Gapdh, 5'-ACC ACA GTC CAT GCC ATC
AC and 5'-TCC ACC ACC CTG TTG CTG TA; Whn, 5'ATG GAG ACC TTG
GGA CTG AC and 5'-TGG CTG AGT GGC ATA GGA GA; pT, 5'-ATC
ACA CTG CTG GTA TAT GGA and 5'-TCA GAG GGG TGG GTA AGA TC;
ß-actin, 5'-GTC GGC CGC TCT AGG CAC CAA and
5'-CTC TTT GAT GTC ACG CAG GAT TTC. PCR amplification for cDNA obtained
from tissues used 25, 26, or 30 cycles, respectively, as indicated for
Gapdh, 28 cycles for Whn and pT,
and 30 cycles for Aire. Amplification of cDNA from sorted
cells used 30 cycles for Gapdh and
ß-actin and 35 cycles for Aire. For
data shown in Fig. 3B, the relative amount of the
first-strand cDNAs produced from each sorted stromal subpopulation was
estimated after amplification of a reference
ß-actin cDNA fragment. PCR products were
separated on a 1.7% agarose gel, visualized by staining with SYBR Gold
(Molecular Probes, Eugene, OR), and images were analyzed using the
QuantityOne gel-doc system (Bio-Rad, Richmond, CA).
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Tissues from 6-wk-old C57BL/6 mice were collected and snap
frozen in liquid nitrogen. Total RNA was isolated using TRI reagent,
and mRNA was isolated from total RNA using the Oligotex kit (Qiagen).
For each sample, 1 µg of mRNA was electrophoresed in a 1%
agarose-formaldehyde gel, transferred to Hybond
N+ membrane (Amersham Pharmacia Biotech, Uppsala,
Sweden) by overnight capillary blotting in 10x SSC. The membrane was
probed with [32-P]-labeled Aire
(54–630 bp; accession no. AJ132243) or Gapdh (566–1017 bp;
accession no. M32599) cDNA fragments. Hybridization was performed for
2 h at 65°C using the QuickHyb buffer (Stratagene, La Jolla, CA)
according to the manufacturer’s instructions. The blot was exposed
overnight using a PhosphoImager Screen (Amersham Pharmacia Biotech),
and results were analyzed by ImageQuant software (Bio-Rad).
In situ hybridization
Sense and antisense digoxigenin-labeled (Roche Molecular Biochemicals) cRNA probes were generated by in vitro transcription using cDNA specific for Aire, Whn, and Rag-1 as templates. ISH was performed as previously reported (40). In short, frozen sections (15 µm) were cut from thymic tissue embedded into OCT, air dried at room temperature (RT; 20 min), fixed in 4% paraformaldehyde (10 min), washed tree times with PBS, and finally acetylated (10 min). Prehybridization was performed overnight at RT with hybridization buffer (50% formamide, 5x SSC, 1x Denhardt’s, 100 µg/ml yeast transfer RNA, 100 µg/ml salmon sperm DNA) using a chamber humidified with 5x SSC. The hybridization mixture was prepared by adding 200 ng of digoxigenin-labeled probe per milliliter of hybridization buffer, which was first heated (85°C, 5 min) and subsequently chilled (4°C). The hybridization mixture was spread over the sections, which were then covered with siliconized coverslips and sealed with rubber cement (Stanford, Bellwood, IL). Slides were placed in Quadriperm dishes (Heraeus Instruments, Hanau, Germany), and hybridization was performed overnight at 68°C in a 5x SSC humidified chamber. Slides were washed initially in 5x SSC at 72°C and subsequently in 0.2x SSC at 70–72°C (60 min) followed by 0.2x SSC at RT (5 min). Next, slides were rinsed with 0.1 M maleic acid, 0.15 M NaCl buffer (pH 7.5), blocked for 1 h in 1% blocking reagent (Roche Molecular Biochemicals) diluted in maleic acid buffer, and incubated for 1 h with alkaline phosphatase-conjugated anti-digoxigenin Ab (Roche Molecular Biochemicals) diluted in blocking solution (1:2500). Slides were washed for 1 x 10 min and 2 x 30 min in equilibrated maleic acid buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.5). For color development, slides were placed upside-down in a Quadriperm dish containing nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Roche Molecular Biochemicals; nitroblue tetrazolium, 112.5 µl; 5-bromo-4-chloro-3-indolyl-phosphate, 87.5 µl; diluted in equilibrated maleic acid buffer). The reaction was performed in the dark at RT and stopped by transferring the slides into PBS containing 1 mM EDTA (10 min). The tissue was counterstained with Methylene Green (0.05% in PBS), and slides were coverslipped using Kaiser’s gelatin (Merck, Darmstadt, Germany). Positive signals are represented by dots of strong purple color, while unspecific signals are detected as small and diffuse dots of weaker intensity. These latter signals are also present in the absence of a specific probe (data not shown).
Immunohistochemistry
Frozen thymic sections (5 µm) were fixed with acetone (Merck) at RT for 15 min and washed in PBS. Endogenous peroxidase activity was blocked by a 15-min incubation with 3% H2O2, 0.1% NaN3 in PBS. The sections were blocked with normal goat serum (10% in PBS) for 30 min. Individual thymic sections were incubated with the appropriate dilution of primary Ab (30 min, RT), washed three times in PBS, and incubated with HRP-conjugated goat anti-rat Ig for 30 min. After additional washing in PBS, the substrate amino-ethyl-carbazole (Sigma) was applied to the sections. The tissues were washed in PBS, counterstained with Mayer’s Hemalaun (Merck), mounted with Crystal/Mount (Biomeda, Foster City, CA), and coverslipped.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To detail the expression pattern of Aire in different
murine tissues, a Northern blot analysis was performed. As shown in
Fig. 1A, specific mRNA was
detected mainly in thymus, and to a lesser extent in spleen and lymph
nodes. At least three distinct bands were consistently observed in each
of these organs, suggesting the existence of multiple Aire
transcripts.
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B). The use of Aire-specific primers with
cDNA from MHC class II+
CD11c+ thymic dendritic cells resulted only in a
very faint amplification product (see below and Fig. 3B).
Thus, stromal cells but not lymphoid cells (at any stage of their
intrathymic development) are responsible for Aire expression
in adult thymic tissue.
Next we determined the pattern of Aire expression during
thymic ontogeny using RT-PCR. For this purpose, RNA was isolated from
thymic tissue of C57BL/6 embryos taken at distinct gestational ages and
analyzed for Aire, Whn, and pT
transcripts. The latter two transcripts were used to monitor the two
dominant cell types present during early thymic ontogeny, i.e.,
epithelial cells and immature thymocytes. Whn is a
transcription factor typically expressed in all thymic epithelial cells
and critical for their growth and differentiation (41, 42). In contrast, pT is a T lymphoid-specific
cell-surface molecule expressed exclusively in early thymocytes, where
it is essential for the maturational transition of a
CD44-CD25+ (TN III) to a
CD44-CD25- (TN IV)
phenotype (43, 44).
Aire-specific cDNA was detected only after E14, while
Whn- and pT-specific transcripts were already
apparent at E12 and E13, respectively. By day 14, commitment to
thymocytes of the TCR ß lineage has already occurred as
demonstrated by the transcription of the pT gene (Fig. 1C and Ref. 45). Moreover, thymic epithelial
subpopulations with a cortical and medullary phenotype can be
distinguished at this point in time (36). Abundant
Aire expression was demonstrated at E16, a time during
thymic development when the formation of DP thymocytes has been
initiated but TCR-mediated thymic selection has not yet begun. The
relative abundance of Aire transcripts decreased somewhat
after day 16 but remained detectable throughout fetal and postnatal
life (Fig. 1C). The observed changes in Whn and
Aire expression during thymic development are likely
accounted for by shifts in the relative frequency of
Aire-positive epithelial cells. Taken together, these
results demonstrate that Aire expression in thymic stromal
cells emerges relatively late during ontogeny but is sustained into
adulthood.
Aire transcription is restricted to a subpopulation of epithelial cells
We next determined the spatial expression of Aire mRNA
in fetal thymi using ISH. In E16 tissue, Aire expression was
detected in small but distinct aggregates scattered throughout the
entire organ (Fig. 2A). In
contrast, ISH using a Whn-specific probe revealed a network
of cells that represented the majority of epithelial cells at this
stage in thymic development (Fig. 2B). Similarly, detection
of a compact network of TEC was also achieved using Abs specific for
cytokeratin 18 (data not shown). Thus, comparison of these two distinct
staining patterns indicates that only a subpopulation of TEC express
Aire-specific transcripts.
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Organization and differentiation of thymic stromal cells into
distinct microenvironments with a typical cellular architecture are
subject to inductive signals provided by thymocytes (6, 11, 46). In particular, the comparison of two mouse strains,
Tg26 and RAGnull mice, has been most
informative in revealing the identity of the thymocyte subpopulation
critical for the formation of a normal thymic microenvironment: TN II
and/or TN III thymocytes provide signals that induce the
three-dimensional organization of TEC (11). Evidence for
this comes from the fact that Tg26 mutant mice, which overexpress
the human CD3 chain in high copy number, display a complete arrest
in early thymocyte development at the transition of TN I to TN II of
intrathymic T cell development, a point in time that corresponds to a
developmental stage before E14.5 of normal thymic organogenesis.
Consequently, the thymic primordium ceases to develop a mature
three-dimensional network of TEC (13). In contrast,
thymopoiesis in RAGnull mutant mice is blocked
later during development, i.e., at the TN III stage, which relates to
E15.5 in the thymic developmental in wild-type mice. The cortical
stroma of RAGnull mice reveals a normal cellular
composition and a typical architectural organization
(13).
To test whether TEC express Aire transcripts before their
formation of a three-dimensional architecture, thymi from Tg26 and
RAGnull mice were analyzed using ISH. At E16 of
development, the pattern of Aire expression in
RAGnull thymi was similar to that seen in
age-matched thymi from wild-type mice (Fig. 2A,
c). In contrast, thymic Aire transcripts could
not be detected in E16 Tg26 thymi despite the ample presence of TEC
(Fig. 2A, e and f). This result was
confirmed by RT-PCR analysis of fetal Tg26 and RAG thymic tissue
(Fig. 2B). In Tg26 thymi, the absence of Aire
expression correlates with the lack of TN II/III thymocytes, while the
presence of Aire transcripts in
RAGnull thymi correlates with the presence of
thymocytes at these specific developmental stages. These results are
consistent with the idea that TN II/III thymocytes induce the
expression of Aire in TEC, which correlates with the
formation of a normal thymic architecture.
Aire is expressed in the medulla and at the corticomedullary junction
To document the spatial expression of Aire transcripts
in a fully differentiated thymic microenvironment, sections of adult
thymi were analyzed by ISH (Fig. 3A). Aire
expression was localized to the medulla and the corticomedullary
junction. While ISH within the medulla revealed a punctate pattern of
staining, the pattern at the corticomedullary junction suggested the
formation of a more continuous arrangement of Aire-positive
epithelial cells. In particular, the slender extensions of the medulla
that protrude into the cortex displayed several layers of
Aire-positive cells, thus forming a concise boundary at the
corticomedullary junction.
Several surface markers have been described that are expressed on
medullary epithelial cells (36, 39, 47, 48, 49, 50). The Ab
designated 29 recognizes a subpopulation of TEC thought to represent
stromal cells bearing features of "activated" cells (36, 51). Otherwise characterized by Ia-specific Abs and lectin UEA-1
binding, this subpopulation of 29+ epithelial
cells has been implicated in tolerance induction (52, 53, 54).
To detect Aire expression in 29+
epithelial cells, stromal cells were isolated by gentle digestion of
thymic fragments. Phenotypically distinct subpopulations were separated
by flow cytometry using a combination of different markers:
CD11c+MHCII+ dendritic
cells; CD11c+29+ dendritic
cells; and CD11c-29+
epithelial cells. cDNAs obtained from these different stromal cell
populations were amplified by semiquantitative PCR to detect
Aire expression. As seen in Fig. 3B, specific
mRNA was preferentially and strongly expressed in the population of
CD11c-29+ epithelial cells
when compared with all other stromal populations tested. Taken
together, 29+ epithelial cells in the medulla and
at the corticomedullary junction are to a large extent responsible for
Aire expression in adult thymic tissue.
To exclude the possibility that the observed absence of Aire
expression in Tg26 (Fig. 2A, e) is simply caused by the
lack of 29+ epithelial cells, Tg26 thymi at
E16 that lack Aire expression were analyzed by
immunohistology for reactivity with either a 29-specific Ab or the
UEA-1 lectin (Fig. 3C and data not shown). Epithelial cells
staining positively for either marker could be easily detected in thymi
of Tg26 mice. These results provide further evidence that the lack
of inductive signals provided by early thymocytes at the TN II/III
phenotype—and not the absence of the responsive epithelial target
cells—account for the deficit of Aire expression in Tg26
thymi.
RelB is essential for Aire expression
Medullary epithelial cells mediate negative selection of developing thymocytes (52) and contribute to late thymocyte maturation (55). For its architectural organization, the medulla is dependent on distinct signals provided by postselection thymocytes (12). These signals may likely be mediated by the transcription factor RelB (36) as mice deficient for RelB display a medullary thymic atrophy (56, 57) and aberrant clonal deletion of autoreactive thymocytes (15). Both traits may be the direct consequence of a severe decrease of Ag 29+, UEA-1+ thymic medullary epithelial cells (36).
To test whether Aire expression can be detected in thymi
devoid of 29+ epithelial cells, mice
homozygous for a null mutation of RelB were analyzed by ISH. The
comparison with wild-type thymi demonstrated that Aire
expression was completely absent in RelBnull
thymi (Fig. 4A, a
and b). Identical results were obtained by use of RT-PCR
(Fig. 4B). Moreover, ISH with a RAG-1-specific
probe revealed a dramatic change in the organization of the thymic
microenvironment: in lieu of a centrally located medulla,
RelBnull thymi displayed multiple small medullary
foci dispersed throughout the RAG+ cortex (data
not shown). Thus, the absence of Aire expression correlated
with a homozygous deficiency for RelB and the disruption of a regular
thymic architecture despite the presence of medullary epithelial cells
other than 29+ stromal cells.
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A normal thymic microenvironment is a prerequisite for generating a repertoire of T cells restricted to self-MHC molecules and tolerant to self-Ags. Therefore, we sought to correlate Aire gene expression with thymocyte selection as Aire has been 1) implicated in the maintenance of tolerance and 2) localized to the anatomical site usually associated with negative selection (58). For this aim, a murine model was analyzed where thymocytes express the 3BBM74 TCR transgene on a RAG-2null background (35). Thymocytes bearing this TCR are positively selected by I-Ab but negatively selected by I-Abm12 (59).
Mice of either the positively selecting or the negatively selecting
haplotype were analyzed by ISH for thymic Aire expression.
In the positively selecting (I-Ab+) thymi,
Aire expression was limited to a few scattered cells located
within the sparse thymic medulla (Fig. 5A). In contrast, abundant
Aire expression at the corticomedullary junction and in the
extended medulla was seen in thymi undergoing negative selection
(I-Abm12+, Fig. 5B). Under conditions
of negative selection, abundant Aire expression may relate
to the TCR-mediated negative selection signals leading to apoptotic
cell death. Alternatively, Aire expression may result from
any form of thymocyte death including death by neglect. To address the
relevance of death by neglect for Aire expression, thymic
tissue from mice deficient for the expression of both MHC class I and
II molecules was analyzed (Fig. 5C). In these
MHCnull mutant mice, almost all thymocytes
undergo cell death by neglect due to the absence of TCR ligands and the
ensuing lack of TCR-mediated survival signals. Thymic tissue from
MHCnull mice revealed Aire expression
restricted to a few scattered cells within the medulla. This pattern
was comparable to the expression noted in mice with only positive
thymic selection. Thus, thymic Aire expression is directly
correlated to the presence of apoptotic cell death due to negative
selection and appears to be independent of mechanisms involved in cell
death by neglect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). The detection of
Aire variants in Northern blots is in keeping with previous
PCR analysis demonstrating alternatively spliced transcripts in
different tissues. In particular, variants with deletions of exon 6, 8,
10, and/or 11, respectively, have been detected among mRNA from thymic
tissue (27, 60). For example, the complete deletion of
exon 10 fails to cause a change in the reading frame but results in a
shortened protein lacking a substantial part of the proline-rich
region; whereas the combined loss of exons 10 and 11 introduces a stop
codon in exon 13 and deletes the second PHD zinc-finger, a motif
present in a number of chromatin-associated transcriptional regulators
(61, 62, 63). It remains to be determined whether all of these
transcripts encode functional proteins with distinct capacities.
Previously, little was known concerning the transcriptional regulation
of Aire. Our experiments now demonstrate that
Aire expression is principally restricted to the
subpopulation of Ag 29+ medullary TEC located
within the medulla and at the corticomedullary junction. During thymic
ontogeny, 29+ stromal cells are first
morphologically detected around E14 (36) and may initially
represent a population of lineage-committed precursors of medullary
epithelial cells. The first Aire-specific transcripts are
detected at E14 during thymic ontogeny (Fig. 1C), in
parallel with the appearance of TN II thymocytes. Two days later, a
network of 29+ medullary stromal cells is
established concomitant with the emergence of more mature thymocytes
and the abundant expression of RelB (36, 64). In adult
mice, the subpopulation of 29+ TEC represent a
network of scattered medullary stromal cells with abundant and
reticulated cell processes. These cells share many membrane markers
known to be critical for Ag presentation to T cells (51)
and have indeed been implicated in negative thymic selection (53, 54).
The cross-talk between developing thymocytes and epithelial cells is
critical for the induction of a typical thymic microenvironment
(6, 11, 46). However, the molecular nature of the signals
that mediate this stromal organization have yet to be defined.
Aire is a possible epithelial target molecule for such a
pivotal interaction as its expression correlates with a normal stromal
organization. The comparison between RAGnull and
Tg26 E16 embryos clearly demonstrates that Aire
expression does not occur in a cell-autonomous manner but is induced
after provision of activation signals mediated by TN II/III thymocytes.
However, the molecular nature of the signals responsible for the
transcriptional regulation of Aire remain presently unknown.
Thus, Aire constitutes to our knowledge the first epithelial
gene product induced by developing early thymocytes and associated with
the correct establishment of a regular thymic microenvironment.
RelB belongs to the NF-
B family of transcription factors that are
characterized by distinct structural features, interaction with each
other and regulation via the IB inhibitor (65). RelB
expression is detected in thymic tissue during embryo development and,
after differentiation into distinct stromal compartments, exclusively
confined to the medulla (64). In RelB-deficient mice, the
outright absence of 29+ thymic stromal cells and
the decreased number of thymic dendritic cells has been correlated with
an irregular medullary architecture (56, 57) and the loss
of efficient negative selection (15, 66). Therefore, our
observation of a complete deficiency of Aire transcripts in
RelBnull mice may be explained by the absence of
29+ medullary stromal cells. In contrast, it is
unlikely that RelB transcription factors constitute a strict
requirement for Aire expression, which, in turn, determines
the fate (e.g., growth and differentiation) of
29+ epithelial cells because Tg26 thymi harbor
29+ epithelial cells but lack Aire
expression. Therefore, the conclusion can be drawn that Aire
expression is not an unconditional requirement for the generation and
maintenance of 29+ medullary epithelial cells.
But it remains to be formally determined whether Aire
transcription is directly RelB dependent. However, the lack of
canonical RelB-binding sequences within the first 600 bp immediately 5'
of the Aire start site suggests that Aire
transcription is independent of this transcription factor
(28).
The molecular properties of the Aire gene product predict a
function as a regulator of gene transcription. In this capacity,
Aire may regulate the architectural organization of the
thymic microenvironment via transcriptional control of downstream
target genes. This notion is supported by our data demonstrating the
complete absence of Aire transcripts and the aberrant
organization of the thymic microenvironment in Tg26 mutant mice. In
these animals, an appropriate corticomedullary differentiation is
missing, epithelial cells are organized in an abnormal two-dimensional
fashion, and the medullary foci are scattered throughout the entire
thymus (11). Importantly, morphological similarities exist
between Tg26- and RelB-deficient mice, as both mutant mice display
scattered and poorly separated areas of medullary epithelial cells
in lieu of a centrally located medulla (36, 56).
Furthermore, in RelBnull mice, the lack of
distinct stromal compartments is reflected by the abnormal spatial
distribution of areas where TCR gene recombination occurs (data not
shown).
Signals transmitted via the TCR/peptide/MHC ligand interaction during later stages of thymocyte development affect the organization of the medullary architecture (46, 67) and determine the selectional fate of developing thymocytes. To correlate Aire expression with thymic selection, RAG-2-deficient mice transgenic for a TCR that is positively selected by I-Ab and negatively selected by I-Abm12 were studied. Both Aire expression and the size of the medullary compartment were strikingly different in the positive (I-Ab) and negative (I-Abm12) selecting backgrounds. In thymi undergoing positive selection, Aire-positive medullary epithelial cells were scattered throughout the organ as either single cells or small densely packed aggregates occasionally located close to the thymic capsule. In contrast, thymi undergoing only negative selection contained a large number of Aire-positive epithelial cells in the medulla and at the corticomedullary junction. Aire expression in epithelial cells is associated with TCR-mediated programmed cell death because there are very few Aire-positive cells in MHCnull thymi, which lack TCR ligands and cause thymocyte death by neglect.
Taken together, the results observed in fetal and adult thymic tissue support the hypothesis that thymic Aire expression is regulated at two distinct steps during thymic ontogeny. First, Aire expression is induced in TEC by the presence of TN II/III thymocytes and confers to the formation of a functional microenvironment capable to effect thymocyte selection. Second, Aire expression by epithelial cells is largely modulated at a later stage of thymic development when DP thymocytes are subjected to selection.
Although the affinity/avidity model of thymic selection predicts that the interaction of the TCR with its peptide/MHC ligand determines the developmental fate of an immature thymocyte, the exact tissue requirements for negative selection are presently a point of discussion. However, ample evidence exists that medullary epithelial cells are capable to delete or anergize self-reactive T cells (52, 54, 68). Here we have demonstrated that Aire expression 1) correlates with the correct structural organization of the thymic microenvironment, 2) is localized to cells and anatomical sites known to effect negative selection, and 3) is modulated by thymocytes undergoing negative selection. Because all of these features are critical for appropriate thymic function, it is conceivable that Aire mutations as observed in APECED patients may affect thymic T cell selection and the formation of self-tolerance.
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Acknowledgments |
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Footnotes |
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2 S.Z. and G.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. G. A. Holländer, Pediatric Immunology, Department of Research, Basel University Medical School, Hebelstrasse 20, 4031 Basel, Switzerland.
4 Abbreviations used in this paper: E, embryonic day; Aire, autoimmune regulator; APECED, autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy; DP, double positive, i.e., CD4+CD8+; ISH, in situ hybridization; pT, pre-TCR -chain; TEC, thymic epithelial cell; TN, triple negative, i.e., CD3-CD4-CD8-; SP, single positive; RT, room temperature.
Received for publication April 5, 2000. Accepted for publication June 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T cell receptor. Science 281:835. gene in development of ß but not 
T
cells. [Published erratum appears in 1995 Nature 378:419.]
Nature 375:795.
B/Rel family. Cell 80:331.[Medline]
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
- dendritic cells but not of lymphoid-related CD8+ dendritic cells. Immunity 9:839.[Medline]
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