Autoimmune regulator (Aire) has been viewed as a central player in the induction of tolerance. This study examines whether Aire can modulate the production of the thymic chemokines involved in corticomedullary migration and thus play a role in intrathymic thymocyte migration and maturation. Aire deficiency resulted in reduced gene expression and protein levels of the CCR4 and CCR7 ligands in whole thymi of mice, as determined by quantitative PCR analysis and ELISA. The expression of the CCR4 ligands coincided with Aire expression in the CD80high medullary thymic epithelial cells, whereas the expression of the CCR7 ligands was detected in other cell populations. Also, the expression pattern of the CCR4 and CCR7 ligands follows that of Aire during postnatal but not during embryonic development. In vitro, overexpression of Aire resulted in an up-regulation of selected CCR4 and CCR7 ligands, which induced selective migration of double-positive and single-positive CD4+ cells. In vivo, Aire deficiency resulted in a diminished emigration of mature CD4+ T cells from the thymi of 5-day-old mice. In conclusion, Aire regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. These results suggest a novel mechanism behind the Aire-dependent induction of central tolerance.
The thymus is the primary lymphoid organ involved in the development of thymocytes and also has an essential role in establishing immune tolerance (1). Impaired clonal deletion or ineffective functional inactivation of self-reactive T cells in the thymic medulla can lead to a breakdown of central tolerance and the development of autoimmune diseases.
An essential role in the regulation of central tolerance has been attributed to the autoimmune regulator (Aire)3 (2). Aire is predominantly expressed in medullary thymic epithelial cells (mTECs) (3) and has several structural features, such as SAND and PHD finger domains, that are characteristic of proteins involved in transcriptional control (2, 4). In humans, mutations in Aire can cause autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy, a syndrome characterized by the presence of Abs to multiple self-Ags and lymphocytic infiltration of endocrine glands, which leads to endocrine autoimmune disorders (5, 6). Similarly, in mice, Aire deficiency results in the development of a variety of autoantibodies and lymphocytic infiltration of multiple tissues (7). In addition, it has been demonstrated that Aire-deficient mice fail to delete organ-specific lymphocytes, thus indicating a defect in negative selection (8).
Aire has been suggested to induce central tolerance by regulating tissue-specific Ag (TSA) expression in the mTECs. mTECs can express thousands of TSAs to the developing thymocytes (9, 10), leading to deletion of self-reactive T cells and, indeed, the purified mTEC population from Aire-deficient mice shows a decreased expression of many (but not all) TSAs (7). However, there is also evidence that impaired expression of TSAs is not the only mechanism behind Aire-induced autoimmunity. For example, Aire-deficient mice have been shown to develop a Sjögren’s syndrome-like autoimmune reaction to α-fodrin, a self-Ag not regulated by Aire (11). Similarly, Aire-deficient nonobese diabetic mice have been shown to develop autoimmune pancreatitis to isomerase A2, another Aire-independent self-Ag (12). Therefore, it is likely that Aire has an additional effect on negative selection independent of its effect on TSA expression (13, 14). The precise mechanism, however, has not yet been characterized.
Thymocyte development, including negative selection, is also dependent on their organized migration through distinct thymic niches, which promotes timely interactions with cortical thymic epithelial cells (cTECs) and mTECs, respectively, and with medullary dendritic cells (reviewed in Ref. 15). The organized migration of thymocytes is orchestrated by the expression of a variety of chemokines in different compartments of the thymic stroma and by stepwise expression of respective chemokine receptors on the developing thymocytes. Thus, the ligands for the chemokine receptors CCR9 and CXCR4 are responsible for the outward relocation of thymocytes mediating the migration of CD4−CD8− double-negative (DN) thymocytes from the corticomedullary junction to the subcapsular area, where they are positively selected (15). The CD4+CD8+ double-positive (DP) thymocytes are then guided through the cortex to the medulla. This corticomedullary migration depends on the expression of two chemokine receptors, CCR4 and CCR7, and their corresponding ligands (15). CCR4 and CCR7 are predominantly expressed on DP and single-positive (SP) CD4+ thymocytes (16, 17, 18), while the ligands for CCR4 and CCR7 are produced predominantly in the thymic medulla (16, 17, 19, 20, 21). Also, the lack of CCR7 has been shown to result in delayed emigration of mature thymocytes (20), suggesting an impaired corticomedullary migration. Since the medulla is believed to be the site of negative selection, the coordinated expression of CCR4 and CCR7 and their corresponding ligands is likely to direct the induction of central tolerance (15). Interestingly, array data from Aire knockout (KO) mice suggest that in addition to down-regulation of TSAs, there is also an aberrant expression of a number of chemokines in the mTEC population (7, 22). Whether Aire itself can regulate the expression of chemokines or has any impact on thymocyte migration has not yet been characterized.
This study aims to clarify whether Aire can regulate thymic chemokine expression and affect the migration of thymocytes. Since CCR4 and CCR7 have been shown to participate in corticomedullary migration and negative selection, we characterize the expression of three ligands for CCR4 (CCL5, CCL17, and CCL22) and two ligands for CCR7 (CCL19 and CCL21). In comparison, we also measure the expression of a chemokine known to mediate the outward relocation of DN thymocytes, the CCR9 ligand CCL25 (15). We ascertain that Aire deficiency results in reduced gene expression and protein levels of both the CCR4 and CCR7 ligands. We show that whereas the CCR4 ligands are induced in the same cell population as Aire, the CCR7 ligands are produced by the adjacent cells. We provide evidence that postnatally, the expression pattern of Aire closely follows that of CCR4 and CCR7 ligands and that overexpression of Aire can induce the expression of thymic chemokines. Finally, we show that overexpression of Aire results in an increased migration of DP and CD4+ thymocytes, whereas lack of Aire results in a delay in mature CD4+ cell emigration.
Materials and Methods
Mice and cell cultures
C57BL/6 mice deficient for the Aire gene were generated at the Walter and Eliza Hall Institute (Melbourne, Australia) via homologous recombination of targeting vectors in mouse C57BL/6 embryonic stem cells (23). Insertion of the Aire targeting vector disrupted exon 8 and brought the LacZ reporter gene under the control of the endogenous Aire promoter, creating an Aire-LacZ fusion. A phosphoglycerate kinase neomycin cassette was used to select positive recombination events and was later removed using the flanking LoxP sites and Cre recombinase. The CCR7-deficient C57BL/6 mice were obtained from The Jackson Laboratory. All mice were bred and maintained at the mouse facility of the Institute of Molecular and Cell Biology (Tartu University, Tartu, Estonia).
The thymic epithelial 1C6 cell line (24) was provided by G. Holländer (University of Basel, Basel. Switzerland) and was cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Life Technologies).
Chemokine expression in whole tissues by real-time quantitative PCR (qPCR)
Thymi and inguinal lymph nodes were dissected from 6- to 8-wk-old wild-type (WT), Aire-heterozygote (Het), and Aire KO mice. RNA was isolated using TRIzol (Invitrogen/Life Technologies) and reverse-transcribed to cDNA using the SuperScript III Reverse Transcriptase (Invitrogen/Life Technologies). qPCR was performed with the Applied Biosystems Prism 7900 SDS instrument using a qPCR SYBR Green Core Kit (Eurogentec) according to the manufacturer’s instructions. The amplification program included an initial denaturation step at 95°C for 10 min, followed by denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min for 40 cycles. SYBR Green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by a melting curve analysis. Every sample was run in three parallel reactions. Primers used to amplify specific gene products from murine cDNA were β2-microglobulin sense, 5′-tgagactgatacatacgcctgca-3′; β2-microglobulin antisense, 5′-gatgcttgatcacatgtctcgatc-3′; K2-8 sense, 5′-aggagctcattccgtagctg-3′; K2–8 antisense, 5′-tctgggatgcagaacatgag-3′; Aire sense, 5′-tcctcaatgagcactcatttgac-3′; Aire antisense, 5′-ccacctgtcatcaggaagag-3′; CCL-5 sense, 5′-gtgcccacgtcaaggagtat-3′; CCL-5 antisense, 5′-cccacttcttctctgggttg-3′; CCL-17 sense, 5′-agtggagtgttccagggatg-3′; CCL-17 antisense, 5′-ccaatctgatggccttcttc-3′; CCL-22 sense, 5′-ctgatgcaggtccctatggt-3′; CCL-22 antisense, 5′-ggagtagcttcttcacccag-3′; CCL-19 sense, 5′-ctgcctcagattatctgccat-3′; CCL-19 antisense, 5′-cttccgcatcattagcaccc-3′; CCL-21 sense, 5′-ccctggacccaaggcagt-3′, CCL-21 antisense, 5′-aggcttagagtgcttccggg-3′; CCL-25 sense, 5′-gtgctgtgagattctacttcc-3′; and CCL-25 antisense, 5′-tatggtttgacttcttcctttcag-3′. The relative gene expression levels were calculated using the comparative Ct (ΔΔCt) method (according to Applied Biosystems), where the relative expression is calculated as 2−ΔΔCt, and where Ct represents the threshold cycle. Except for the analysis of Aire and chemokines in developmental dynamics (see below), the data was normalized to the expression level of the β2-microglobulin gene.
Chemokine protein levels in whole thymi by ELISA
Thymi from 6- to 8-wk-old WT or Aire KO mice were dissected and collected into RPMI 1640. Small cuts were made into the capsules of thymi and the thymocytes were released by repetitive pipetting. The remaining thymic fragments were incubated in 0.5 mg/ml dispase/collagenase (Roche) and 5 μg/ml DNase I (AppliChem) in PBS at 37°C for 20 min with gentle agitation. The released cells were collected to separate fractions and fresh enzyme solution was added four times. Each cell fraction was counted and the final two or three digested fractions were pooled. A negative depletion was performed to enrich for CD45− cells using CD45 microbeads (Miltenyi Biotec) and the AutoMACS system (Miltenyi Biotec) per the manufacturer’s instructions. The negative fraction was stained with anti-G8.8-FITC (anti-EpCAM, generated from a G8.8 hybridoma cell line), anti-Ly51-PE (BD Biosciences), anti-CD45-PerCP-Cy5.5 (BD Biosciences), and anti-CD80 biotin (BD Biosciences) followed by second-stage staining with streptavidin-PE-Cy7 (Serotec). Cell sorting and analysis were performed on a FACSAria (BD Biosciences) instrument to get the fractions of mTECs (CD45−, G8.8high, Ly51low) and cTECs (CD45−, G8.8low, Ly51high). According to the CD80 expression, the mTEC fraction was further divided into the CD80high and CD80low mTECs. Thereafter, the RNA was purified by using a RNeasy Micro Kit (Qiagen) followed by reverse transcription and qPCR as described above.
Embryonic (E13.5, E15.5, and E17.5), newborn, neonatal day 10 (10 day) and adult (6-wk, 6-mo) WT mouse thymi were dissected and used in developmental dynamics analysis. RNA was isolated using TRIzol followed by reverse transcription and qPCR as described above. To overcome variations in the cellular composition during development and to detect the expression of Aire and chemokines in the epithelial cell population only, the data were normalized to the expression level of keratin 8 mRNA, which is expressed selectively by thymic epithelial cells, shows an equal expression level in the mTECs and cTECs, and is not influenced by Aire gene expression (7, 22).
An adenoviral expression system (AdAire-GFP vs Ad-GFP) was used as previously described (25). TEC 1C6 cells were infected at ∼70% confluence with Ad-Aire-GFP or Ad-GFP in 500 μl of serum-free OptiMEM (containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) for 1 h. The medium was replaced thereafter with standard medium, and the cells were further incubated for 48 h. The cells were then collected using trypsin-EDTA and assessed for infection rate by quantifying the percentage of GFP-positive cells in Ad-Aire-GFP- or Ad-GFP-infected TECs by a FACSCalibur (BD Biosciences). RNA was isolated from the cells using TRIzol followed by reverse transcription and qPCR as described above.
Thymocyte chemotaxis in vitro
Thymocyte chemotaxis was assayed in Transwell inserts (Corning) with a 5-μm pore diameter. Thymocytes were obtained from thymi of 6- to 8-wk-old WT C57BL/6 mice. Small cuts were made into the capsules of thymi and the thymocytes were released by repetitive pipetting. The collected thymocytes were resuspended in standard TEC 1C6 medium at 5 × 107+ cells further subdivided as DN (CD4−CD8−), SPCD4 (CD4+CD8−), SPCD8 (CD4−CD8+), and DP (CD4+CD8+) thymocytes.
Emigration of mature T cells ex vivo and in vivo
The functional effect of Aire deficiency on thymocyte maturation/emigration was studied using newborn thymus organ culture as previously described (20, 26), with some modifications. Whole thymi were freshly dissected from 5-day-old WT, Aire KO, or CCR7 KO (positive control) mice and placed at the medium/air interface in the Transwell inserts (Corning) with a 5-μm pore diameter. The thymi were incubated at 37°C/5% CO2 and, 16 h later, the thymi as well as the emigrated cells were collected. The emigrated thymocytes as well as the thymocytes that remained in the thymus were counted and stained for CD45, CD4, and CD8 expression (see above). Mature thymocyte emigration was calculated as percentage of emigration by taking the ratio of emigrated SP cells and remaining SP cells. To further characterize the emigration of mature thymocytes in 5-day-old WT vs Aire KO mice, the cells were taken from both sides of the membrane (i.e., migrated vs unmigrated thymocytes) and costained with anti-CD45-allophycocyanin, anti-TCRβ-PE-Cy5 (both from BD Biosciences), anti-CD69-FITC, and anti-Qa2-PE (both from Abcam). The emigration of CD4+TCRβ+CD69−Qa2+ cells was calculated as described above.
Also, the emigration of mature T cells was studied by counting the number of T cells in spleens from 5-day-old WT, Aire KO, or CCR7 KO mice (20). The dissected spleens were digested with dispase/collagenase to get a single-cell suspension. The cells were counted thereafter and stained for CD45, CD4, and CD8 (see above). The number of mature, emigrated T cells was calculated as the total number of SP cells per animal.
Expression of Aire and thymic chemokines in thymi and lymph nodes
To determine whether Aire can affect thymic chemokine expression at the whole tissue level, we selected three previously characterized CCR4 ligands (CCL5, CCL17, and CCL22) and two previously characterized CCR7 ligands (CCL19 and CCL21) as well as the ligand for CCR9 (CCL25) for investigation. We measured the expression level of these transcripts in whole thymi and lymph nodes from WT, Aire Het, and Aire KO mice. qPCR analysis showed a decreased expression of all CCR4 and CCR7 ligands in the thymi of Aire KO mice compared with WT mice (Fig. 1⇓A). We did not, however, observe an allelic dose dependency of the Aire gene for the CCR4 and CCR7 ligands as there was no significant difference between the WT and Aire Het mice (data not shown). In contrast to the CCR4 and CCR7, the expression of the CCR9 ligand CCL25 was increased in the Aire KO mice compared with the WT mice as measured at the whole tissue level (Fig. 1⇓A).
In contrast to the thymi, expression of the measured chemokines in lymph nodes was not affected by the expression level of Aire (Fig. 1⇑A).
To determine whether the changes in gene expression mirror the changes at the protein level, we also measured the levels of CCR4 and CCR7 ligands in whole thymic tissues from WT and Aire KO mice by ELISA (Fig. 1⇑B). We found significantly reduced levels of the CCR4 ligands CCL5 and CCL22 as well as the CCR7 ligands CCL19 and CCL21 in whole thymi from Aire KO mice compared with the WT mice, whereas the level of CCL17, although slightly reduced in the Aire KO mice, was not significantly decreased (Fig. 1⇑B).
Coexpression of Aire and CCR4 ligands in CD80high mTECs
To determine whether the changes in chemokine expression reflect the changes in the same cell subpopulation in which Aire is expressed, we FACS sorted the cTECs and mTECs and further divided the latter into the CD80low and CD80high subpopulations (Fig. 2⇓A). The expression levels of Aire, as well as the expression levels of all studied chemokines, were found to be remarkably higher in the mTEC population compared with the cTECs (Fig. 2⇓B). Within the mTECs, the expression of Aire as well as the expression of the CCR4 ligands CCL5, CCL17, and CCL22 was more prominent in the CD80high population of WT mice and down-regulated in the corresponding subpopulation of the Aire KO mice. Regarding the CCR7 ligands, CCL19, although also showing the highest expression in the CD80high subpopulation of WT mTECs, was not significantly down-regulated in the same subpopulation from Aire KO mice, whereas CCL21, although down-regulated in Aire KO mice, was preferentially expressed in the CD80low subpopulation of mTECs. The expression of the CCR9 ligand CCL25 was specifically up-regulated in the CD80high mTECs of the Aire KO mice.
Expression of Aire and thymic chemokines in the ontogeny of WT mice
If the expression of thymic chemokines is dependent on Aire, this should be evident throughout the development of thymic tissue. However, the thymic cellular content and volume change significantly during development. To limit our analysis to the epithelial cell subsets only, we normalized our data to the keratin 8 gene, which is expressed independently of Aire by medullary and cortical epithelial cells. Thymi from various embryonic, neonatal, young, or adult developmental stages were analyzed for Aire and thymic chemokine expression (Fig. 3⇓). Expression of the majority of chemokines, including CCL5, CCL17, CCL21, and CCL25, was already detectable at the very earliest time point studied, E13.5, while Aire showed little if any expression at this point. After birth, however, the expression level of most of the thymic chemokines followed the pattern of Aire, peaking at day 10 and gradually decreasing thereafter. Again, the only exception was the CCR9 ligand CCL25, whose expression started very high at E13.5, decreased gradually to E17.5, and reached a second peak at day 10, one which lasted until 6 wk of age (Fig. 3⇓).
Overexpression of Aire increases thymic chemokine expression
Next, we aimed to show that Aire, as a single factor, is sufficient to up-regulate thymic chemokine expression. We used an adenoviral expression system (AdAire-GFP vs Ad-GFP) to determine whether the specific overexpression of Aire has an effect on the expression of chemokines. Incubation with AdAire-GFP or Ad-GFP viruses resulted in a virtually equal infection rate as determined using FACS by evaluating the percentage of GFP-positive cells at 48 h (AIRE-GFP: 38.1 ± 4.3% vs ad-GFP: 42.8 ± 2.8%; mean with SEM; n = 3). However, compared with Ad-GFP, the AdAire-GFP infection resulted in an increased expression of the CCR4 ligands CCL5 and CCL22, the CCR7 ligand CCL19, as well as the well-characterized Aire-regulated gene Insulin 2, whereas there was no change in the expression of CCL17, CCL21, or CCL25 (Fig. 4⇓). These results demonstrate that even in the absence of signals from other cell types normally present in thymus, Aire expression is sufficient to induce the expression of a number of thymic chemokines in thymic epithelial cells.
Overexpression of Aire induces DP and SPCD4 thymocyte migration
Since overexpression of Aire resulted in an up-regulation of the CCR4 and CCR7 ligands, we wanted to clarify whether this overexpression can also result in the selective migration of thymocytes toward the Ad-Aire-GFP-infected thymic epithelial cells. In this setting, infection with Ad-GFP did not have any effect on the total numbers of thymocytes that migrated (negative control: 0.084 ± 0.01 × 106 vs Ad.-GFP: 0.080 ± 0.01 × 106; Student’s t test, p > 0.05; n = 5), whereas overexpression of Aire resulted in a significant increase in migrated thymocyte numbers (Ad-Aire-GFP: 0.124 ± 0.01 × 106, p < 0.05 compared with Ad-GFP). This increase in thymocyte migration was specific for the DP and SPCD4 cells, i.e., the cells expressing CCR4 and CCR7 (Fig. 5⇓, A and B). To characterize the magnitude of the effect of Aire overexpression in this setting, we also used cell supernatants from TNF-α-stimulated thymic epithelial cells and evaluated the effect on thymocyte chemotaxis. Supernatants from TNF-α-treated cells induced a significant increase in DP thymocyte chemotaxis, which was in the same order of magnitude as the effect induced by Ad-Aire-GFP. However, the supernatants from TNF-α-treated cells had no effect on chemotaxis of other cell subsets. CCL19 alone, on the other hand, resulted in a selective induction of SPCD4 and SPCD8 chemotaxis (Fig. 5⇓B).
Aire-induced thymocyte migration is inhibited by Abs against CCR4 and CCR7 ligands
Consequently, we aimed to clarify whether the effect of Aire overexpression on DP and SPCD4 chemotaxis is mediated by an increased release of CCR4 and CCR7 ligands. We used Ad-Aire-GFP-infected cell supernatants along with combinations of Abs to all known CCR4 and CCR7 ligands and characterized the migration of DP and SPCD4 thymocytes. The effect of Aire on SPCD4 migration was inhibited by the presence of Abs against CCR7 but not CCR4 ligands (Fig. 5⇑C). However, the effect of Aire on DP cell migration was inhibited by both the CCR4 and the CCR7 ligand Abs. The combination of CCR4 and CCR7 ligand Abs did not produce any further effect when compared with CCR4 or CCR7 ligand Abs alone (Fig. 5⇑C).
Aire deficiency results in delayed emigration of mature thymocytes
To determine whether the lack of Aire has also an impact on migration of thymocytes in vivo, we used a previously characterized model of thymocyte emigration that should reflect the corticomedullary migration and maturation of early thymocytes. Using this model, it has been shown that in CCR7 KO mice, the impaired corticomedullary migration of thymocytes results in delayed emigration of mature SP thymocytes, which can be detected only during the neonatal period (20). In this study, we used the thymi from 5-day-old WT, Aire KO, and CCR7 KO (positive control) mice and calculated the number of emigrated mature SP cells. We observed that the number of emigrated SPCD4 cells, as well as total number of SP cells, was significantly reduced in the 5-day- old Aire KO thymi compared with WT thymi (Fig. 6⇓, A and B). In the CCR7 KO mice, the numbers of emigrated SPCD4, SPCD8, as well as the total number of emigrated SP cells were all reduced compared with the WT mice. In parallel, we characterized the maturation and emigration of early thymocytes by quantifying the number of mature T cells in the spleen of WT, Aire KO, and CCR7 KO mice at 5 days. We found that the number of both CD4+ and total T cells was reduced in the Aire KO mice, whereas in the CCR7 KO mice the numbers of CD4+, CD8+, and the total T cells were all reduced (Fig. 6⇓C).
Because Aire deficiency has been shown to result in reduced numbers of the SPCD4+ of the latest developmental stage (i.e., TCRβ+CD69−Qa2+) (27), we also aimed to characterize whether this difference can be detected in the emigration assay. However, by using thymi from 5-day-old mice, we could not detect any significant difference in emigration of the CD4+TCRβ+CD69−Qa2+ thymocytes between the WT and Aire KO mice (percent emigration mean ± SEM; WT: 21.2 ± 11.7, Aire KO: 17.3 ± 4.7; Student’s t test, p > 0.05; n = 5–6).
Coordinated migration of thymocytes through distinct compartments of thymus is a prerequisite for controlled T cell development. The migration of thymocytes from the corticomedullary junction to the cortex and from the cortex to the medulla is coordinated on the one hand by stepwise expression of chemokine receptors on the thymocytes and, on the other hand, by expression of different chemokines by different thymic cellular compartments (15). In this study, we show that in Aire-deficient mice there is a down-regulation of the two major groups of chemokines responsible for corticomedullary migration: the ligands for CCR4 and CCR7. Our results thus point to a novel mechanism by which Aire may affect the induction of central tolerance. On the other hand, the expression of the chemokine known to mediate the outward relocation of DN thymocytes, CCL25 (28), is up-regulated in the Aire KO mice, suggesting that the coordinated migration of thymocytes in Aire-deficient mice may be influenced at different levels of maturation.
The effect of Aire deficiency on chemokine expression has been previously suggested by Anderson et al. (8) using gene chip analysis of mTECs from WT and Aire KO mice. In agreement with the previous report, the present study shows a reduction of CCL17 and CCL22 as well as an increase in CCL25 in the Aire-deficient mice. However, as opposed to the previous article, we show also reduced levels of CCL5, CCL19, and CCL21 in the Aire KO thymi. These differences are likely to be due to the higher sensitivity of the qPCR method, used in this study, or possibly due to the fact that whole thymi rather than sorted mTECs were used.
In addition to the thymus, lymph nodes have recently been reported to express Aire, and it has been suggested that this peripheral Aire can also contribute to the induction of tolerance (29, 30). In this study, we show that at least at the whole tissue level, the majority of chemokines are not affected by Aire-deficient lymph nodes. This finding, in agreement with a previous report, suggests that the number of target genes for Aire is different and more limited in the periphery (30) and also indicates a minor role for Aire in the regulation of peripheral chemokines.
The expression of Aire in the thymus is confined to a specific subset of epithelial cells, the CD80high mTECs (22). In this study, we show that the expression and regulation of all CCR4 ligands is indeed localized to the same subset of cells, suggesting that the effect of Aire on CCR4 ligand induction is located in, although not certainly limited to, the same cell population. The expression of the CCR7 ligands, on the other hand, is either not localized to the CD80high mTECs (CCL21) or is not regulated by Aire in this cell population (CCL19), which indicates an indirect effect of Aire on CCR7 ligand induction. Whereas the CD80low mTEC seems to be the target cell for CCL21 production, the source for CCL19 remains to be clarified. However, the likely candidates include thymic dendritic cells and fibroblasts, both of which are known to produce considerable amounts of the CCR7 ligands (31).
Aire has recently been associated with changes affecting also the Aire-negative subset of mTECs. Hence, the Aire-deficient thymi have an altered expression of several transcription factors associated with multipotentiality (32), have a decreased expression of CD80 and MHC class II in mTECs noncommitted to express Aire (33), and an altered ultrastructure of the undifferentiated mTECs (34). In line with these findings is the current data showing an altered expression of chemokines outside the CD80high mTECs of Aire KO mice and collectively suggesting an effect for Aire beyond the end-stage fully mature mTECs.
To determine whether the thymic expression of Aire and chemokines follow the same pattern during mouse development, we monitored their expression from E13.5 to 6 mo. We detected a clear signal for many chemokines at E13.5, before the obvious increase in Aire signal at day E15.5, which suggests that in early development, mechanisms other than Aire trigger the chemokine expression needed for arrival of early thymocytes. However, during postnatal development and involution, both the CCR4 and the CCR7 ligands followed the expression pattern of Aire by peaking at day 10 and gradually declining thereafter. Thus, these data are compatible with Aire playing a role as a postnatal contributor to the regulation of chemokines behind corticomedullary thymocyte migration.
We further characterized Aire-dependent chemokine expression by adenoviral experiments inducing Aire expression in thymic epithelial cells. We established that overexpression of Aire as a single factor is sufficient to induce the expression of the CCR4 ligands CCL5 and CCL22 as well as the CCR7 ligand CCL19. In contrast, CCL17 and CCL21, although clearly Aire dependent in other experimental settings, were not induced, suggesting that optimal chemokine induction requires additional signals or cell types. Likewise, the CCR9 ligand CCL25, although negatively regulated in an Aire-deficient situation, was not reduced by Aire overexpression, suggesting again an indirect mechanism behind this effect.
As chemokine receptors are differentially expressed during different stages of thymocyte development, we also tested whether an increase in Aire-induced chemokine expression results in a significant and selective increase in thymocyte migration by using supernatants from cells overexpressing Aire. In this chemotaxis assay, we showed that induction of Aire can selectively induce migration of DP and SPCD4 thymocytes. The fact that these cell populations are the ones selectively expressing CCR4 and CCR7 and naturally migrating from cortex toward medulla (16, 17, 18), as well as the fact that the Aire-induced migration of these cells was inhibited by the presence of CCR4 and CCR7 ligand Abs, further strengthens the idea that Aire is a contributor to the corticomedullary migration of thymocytes.
Finally, we aimed to determine whether the lack of Aire results in significant changes to corticomedullary migration of thymocytes in vivo. We used an experimental setting where the functional significance of CCR7 deficiency has previously been described (21). We showed that similar to the case in CCR7 KO mice, there is a delay of emigration of mature lymphocytes in the Aire KO mice. Interestingly, we see a significant delay of emigration for the CD4+ cells but not for the CD8+ cells, perhaps reflecting a predominant expression of CCR4 and CCR7 on SPCD4 thymocytes.
A delayed emigration of thymocytes is not the only similarity between the Aire-deficient and CCR7 KO mice. Just like the Aire KO mice, CCR7-deficient mice are also characterized by lymphocytic infiltrations in multiple tissues, including the lachrymal gland, stomach, lung, salivary gland, pancreas, and liver; they are also characterized by the presence of multiple autoantibodies, including the ones against α-fodrin (35). In addition, CCR7-deficient mice are characterized by altered thymic microarchitecture, illustrated by smaller but more numerous medullary areas, which are sometimes misplaced under the cortical rim (36). Interestingly, recent publications have also shown an altered differentiation program in the mTECs of Aire KO mice (33, 37), further stressing the similarities between Aire-deficient and CCR7-deficient mice. Together, these similarities suggest the involvement of a common mechanism for the ligands of CCR7 and Aire.
Also, a recent study has shown a severe reduction of CD80high mTECs in H2-Aa−/− mice (38), indicating a requirement for SPCD4 thymocytes in thymic medulla maturation and proliferation. It remains to be determined whether the impaired corticomedullary migration of thymocytes or delayed maturation of SPCD4 cells described in our study contributes to the postponed functional maturation of thymic medulla or changes in mTEC differentiation. Another study has provided evidence that the final maturation of SP thymocytes might be blocked in the Aire KO mice (27). However, we do not see any difference in early emigration of CD4+TCRβ+CD69−Qa2+ cells between Aire KO and WT mice suggesting that mechanisms other than impaired corticomedullary migration might mediate this effect.
In conclusion, we show that Aire can regulate the mRNA and protein expression of the CCR4 and CCR7 ligands in whole thymi. The CCR4 ligands are regulated by Aire in the CD80high mTECs, whereas the CCR7 ligands are up-regulated in adjacent cells. Overexpression of Aire as a single factor induces the expression of CCL5, CCL22, and CCL19 and results in an increase in DP and SPCD4 thymocyte migration, an effect inhibited by the CCR4 and CCR7 ligand Abs. Also, the lack of Aire in vivo results in a delayed emigration of mature thymocytes. These data suggest that regulation of thymic chemokines and corticomedullary migration of thymocytes contribute to the Aire-dependent induction of central tolerance.
We thank all past and present laboratory and support staff in the laboratories of H.S.S. and P.P.
The authors have no financial conflict of interest.
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↵1 This work was supported by Eurothymaide and EURAPS, 6th Frame Program of the European Union (to P.P. and H.S.S.), by a Wellcome Trust senior fellowship grant (to P.P.), by National Health and Medical Research Council Fellowships 171601 and 461204, by National Health and Medical Research Council Program Grants 257501, 264573, and 406700 (to H.S.S.), by Estonian Science Foundation Grants 7559, 7197, and 6663 (to M.L., K.K., and P.P.), by the European Regional Fund and Archimedes Foundation (to M.L., K.K., and P.P.), and by Estonian Targeted Financiation Grant SF0180021s07 (to P.P.).
↵2 Address correspondence and reprint requests to Dr. Martti Laan, Molecular Pathology, Biomedicum, Ravila 19, Tartu 50414, Estonia. E-mail address:
↵3 Abbreviations used in this paper: Aire, autoimmune regulator; mTEC, medullary thymic epithelial cell; cTEC, cortical thymic epithelial cell; DN, double negative; DP, double positive; SP, single positive; TSA, tissue-specific Ag; KO, knockout; qPCR, quantitative PCR; WT, wild type; Het, heterozygote; Ct, threshold cycle.
- Received December 9, 2008.
- Accepted October 10, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.