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Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire

Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire+ and Aire− MEC subsets through flow cytometric analysis of 5-bromo-2′deoxyuridine (BrdU) incorporation. The Aire+ MEC subset was almost entirely postmitotic and derived from cycling Aire− precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire− MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire+ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a “terminal differentiation” model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire+ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.

To guard against autoimmunity, the thymus imposes self-tolerance on diff erentiating thymocytes. For conventional ␣ / ␤ T cells, this function is performed primarily by dendritic cells and thymic epithelial cells (TECs). It has emerged that TECs ectopically express a wide array of peripheral-tissue antigens (PTAs), a representation of self that substantially expands the scope of central tolerance ( 1 ). This promiscuous gene expression is compromised in humans and mice lacking the autoimmune regulator (AIRE; Aire in mice), leading to autoimmunity that targets a range of organs and tissues. Aire ' s tolerogenic function acts via medullary epithelial cells (MECs), because loss of the protein in these cells alone is necessary and suffi cient to cause autoimmunity ( 2 ). How this rare cell population comes to express such a large and heterogeneous array of PTAs, how it manages to eff ectively purge the enormous repertoire of maturing thymocytes, and what implications this vast ectopic expression of proteins has for its own biology remain open questions.
Two models, both based on familiar paradigms in the fi eld of developmental biology, have been proposed to explain Aire ' s function in MECs. The " terminal diff erentiation " model is rooted in the fi nding that a hierarchy of promiscuous gene expression exists among TEC subsets ( 3 ). It is postulated that increasingly promiscuous expression correlates with MEC diff erentiation and the CD80 hi , MHC II hi subset of MECs (MEC hi ), which expresses Aire and the most PTA genes, representing the most mature cell type ( 1 ). Two predictions of this model are that, among TECs, individual MEC hi express the most diverse array of PTAs, and that these cells are postmitotic products that ultimately perish (i.e., they are terminally differentiated; Fig. 1 ).
The competing " developmental " or " progressive restriction " model posits that Aire expression and promiscuous gene transcription are properties of immature precursor TECs ( 4 ). According to this scenario, Aire drives the diff erentiation of MECs into progressively restricted cell fates that recapitulate the transcriptional programs of diff erent epithelial lineages. Therefore, it is predicted that the transcripts present in an individual mature MEC should refl ect one such program. Furthermore, the Aire ϩ MECs should be an immature, cycling cell type ( Fig. 1 ).
One of the key distinguishing features of the two models is the diff erentiation state of Aire ϩ MECs. The recent description of a precursor of Aire ϩ MECs in the fetal thymus indicated that Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire ؉ and Aire ؊ MEC subsets through fl ow cytometric analysis of 5-bromo-2 deoxyuridine (BrdU) incorporation. The Aire ؉ MEC subset was almost entirely postmitotic and derived from cycling Aire ؊ precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire ؊ MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire ؉ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a " terminal differentiation " model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire ؉ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.
Aire ϩ MEC hi were not distinguished from Aire Ϫ MEC hi by expression of the MEC cell-surface markers, Ulex europeaus agglutinin 1 (UEA-1; Fig. 2 C ) and CD80 (not depicted), nor by expression of a range of other markers previously associated with precursor cell types, including CD24, CD44, K5, K8, or Sca-1 (not depicted). However, both the Aire ϩ and Aire Ϫ MEC hi subsets expressed CD86 and programmed death 1 ligand (PD-L1) at variable levels ( Fig. 2 C ), indicating further heterogeneity among MEC hi and the potential to provide distinct co-stimulation signals to thymocytes. Nonetheless, we found the phenotypes of Aire ϩ and Aire Ϫ MEC hi to be very similar overall.
Aire ؉ MECs are postmitotic The proliferative status of Aire ϩ MECs is important for determining whether the terminal diff erentiation or progressive restriction model better describes MEC diff erentiation. A terminally diff erentiated population would be postmitotic, whereas, conversely, a precursor MEC population would rely on division for maintenance and phenotypic diversifi cation. Previous studies showed that the adult MEC hi subset was more proliferative than CECs and MEC lo , according to assessments of DNA content ( 6 ), Ki67 expression ( 7 ), and BrdU labeling ( 7 ); however, the Aire ϩ MEC hi component was not specifi cally analyzed. On the other hand, relying on immunohistology, Hamazaki et al. found that adult Aire ϩ MECs did not incorporate BrdU 4 h after injection ( 5 ), but this technique precluded quantitative analysis, and it was not clear whether this time point labeled suffi cient MECs for detection. In this study, we optimized fl ow cytometric analysis of TECs for concurrent detection of BrdU and Aire to assess proliferation of Aire + and Aire Ϫ TEC subsets, and we also followed the fl ow of BrdU label with time to investigate their population dynamics.
First, to assess the relative levels of cell division among TEC subsets, we injected mice with a single dose of BrdU, waited 12h, stained enriched suspensions of thymic stromal cells with antibodies to TEC markers and BrdU, and analyzed them by fl ow cytomety. A substantial proportion of TECs had incorporated BrdU 12 h after injection ( Fig. 3 A ). In accordance with previous studies ( 6,7 ), there was considerably lower incorporation by the MEC lo compared with the MEC hi population (average of 7.1 Ϯ 2.9% vs. 15 Ϯ 2.7%, respectively). Within the MEC hi subset, the Aire Ϫ population had a surprisingly high level of BrdU incorporation, with 27% of the cells in this subset dividing during the pulse ( Fig. 3 A ). In contrast, minimal BrdU incorporation was detected in the Aire ϩ MEC hi subset, indicating that few, if any, Aire ϩ cells were cycling ( Fig. 3 A ). Second, to follow the short-term fate of cells that divided during the BrdU pulse, we measured BrdU retention by TECs at various time points after injection. Within the whole TEC population, labeled cells diminished by one third within the fi rst 3 d, then stabilized for the next 2 d ( Fig. 3 B , far left). The pattern of decay was similar in the MEC lo and Aire Ϫ MEC hi subsets at magnitudes refl ecting their initial BrdU incorporation ( Fig. 3 B , left and right). this subset is a downstream product in the MEC lineage ( 5 ); however, it remains to be determined how far downstream this is and whether the same sequence of diff erentiation holds for the steady-state adult thymus. In contrast, evidence of a high rate of cell division for the adult MEC hi population ( 6, 7 ), the subset with the highest Aire levels, was interpreted to support the notion that these cells represent cycling precursors ( 8 ).
In this paper, we have exploited fl ow cytometric analysis of BrdU incorporation to provide a cell-by-cell view of the dynamics of the various MEC populations in adult mice. The data obtained support the terminal diff erentiation model and argue that Aire may not only drive expression of PTA but also promotes cellular changes to enhance their cross-presentation.

RESULTS AND DISCUSSION Phenotypic characterization of Aire ؉ MECs
The Aire gene is transcribed predominantly in the MEC hi subset ( 3, 7 ), but it was not known how homogeneously these cells express the Aire protein. Therefore, we performed fl ow cytometric analysis of adult thymic stroma using an mAb specifi c for Aire ( Fig. 2 A ) ( 9 ). Intracellular staining of enriched TEC suspensions from aire Ϫ / Ϫ mice gave no signal but revealed a subset of Aire ϩ cells composing 50 -60% of the MEC hi population in aire ϩ/ϩ mice ( Fig. 2 B ). A very low proportion of MEC lo were Aire ϩ , whereas no staining above background was detected in cortical epithelial cell (CECs; Fig. 2 B ). These values translated to an average of 8.4 ϫ 10 4 ( Ϯ 2.5 ϫ 10 4 ) Aire ϩ MECs in the thymus of a 6-wk-old mouse.  1) proposes that TEC precursors are Aire Ϫ , cycling cells expressing few PTAs that give rise to mature, Aire ϩ , CD80 hi , MHC II hi MECs that express the greatest diversity of PTAs. Conversely, the progressive restriction model (model 2) predicts that precursor TECs are Aire ϩ , cycling cells that express the greatest range of PTAs and differentiate down specifi c lineages into mature TECs expressing PTAs of terminally differentiated cells.

BRIEF DEFINITIVE REPORT
constant access to label. The proportion of cycling cells at the outset of incorporation was assessed 2 h after BrdU injection. Again, the Aire Ϫ MEC hi population incorporated the most BrdU and Aire ϩ MEC hi only background levels ( Fig. 3 C , right and far right), confi rming the data in Fig. 3 A , which used a longer pulse. Interestingly, almost all Aire Ϫ MEC hi were BrdU ϩ by 5.5 d ( Fig. 3 C , right), indicating that this In contrast, the proportion of BrdU ϩ cells in the Aire ϩ MEC hi population increased over the fi rst 3 d after the pulse, then diminished ( Fig. 3 B , far right). Given that Aire ϩ MEC hi were not cycling, the entry of BrdU into this compartment must be derived from cycling Aire Ϫ precursors.
To assess the turnover of these populations, we analyzed the kinetics of BrdU incorporation over a 2-wk period of A lag period of 36 h before BrdU ϩ cells accumulated in the Aire ϩ MEC hi population ( Fig. 3 C , far right) refl ected the precursor/product relationship observed in the decay kinetics and represents the minimum amount of time such a precursor takes to up-regulate Aire after cell division. Taking this delay into account, we estimate that an average of 13% ( Ϯ 2.1%) of this subset is replaced each day.
The slow turnover of MEC lo and their expression of the putative TEC precursor marker, MTS-24 ( 10 ), are reminiscent subpopulation is composed entirely of rapidly dividing cells with a prodigious replacement rate of 20% per day. In contrast, the TEC, MEC lo , and Aire ϩ MEC hi subsets showed bimodal incorporation curves, with saturation of a rapidly dividing component ( ‫ف‬ 60% of each) within 6 d, followed by a reduced rate of incorporation by a component with a lower level of proliferation ( Fig. 3 C , far left, left, and right). This result indicates that mixtures of cells with both high and low turnover rates maintain these subsets.

BRIEF DEFINITIVE REPORT
The fi nding that Aire ϩ MEC hi are postmitotic and have a high turnover rate raises the question of whether Aire itself causes proliferative arrest and/or apoptosis of MEC. This notion was supported by the fi nding that aire Ϫ / Ϫ mice have an increased proportion and number of MEC hi ( Fig. 5 A, dot  plots) ( 2 ) at the expense of the MEC lo subset ( Fig. 5 A , right). To determine whether this increase was caused by greater proliferation of MEC hi in the absence of Aire, we injected BrdU into aire ϩ/ϩ and aire Ϫ / Ϫ mice and analyzed incorporation by MEC hi 12 h later. Surprisingly, the proportion of BrdU ϩ MEC hi in aire Ϫ / Ϫ mice was signifi cantly lower than in aire ϩ/ϩ controls, suggesting that the higher numbers of MEC hi in aire Ϫ / Ϫ mice are not derived from increased proliferation but stem from expansion of postmitotic MEC hi (i.e., analogous to Aire ϩ MEC hi ; Fig. 5 B ). Reduced Aire levels in aire ϩ/ Ϫ mice ( 9 ) did not impinge on the MEC hi proportion, numbers, or BrdU incorporation (unpublished data). Therefore, Aire does not directly inhibit MEC hi proliferation; rather, it modulates the overall number of the postmitotic population in some other way, perhaps by inducing apoptosis.
Studies of apoptosis in TECs ex vivo were not feasible because of the induction of high levels of Annexin V labeling and caspase activation by the thymus digestion procedure (unpublished data). Staining of thymus sections with the activated caspase marker VAD-FMK demonstrated in situ apoptosis of thymocytes but did not label Aire ϩ or Aire Ϫ K5 ϩ TECs. This presumably refl ects the fact that thymic macrophages and dendritic cells promptly phagocytose dying cells, as they do the vast majority of thymocytes that perish daily but escape detection by apoptosis assays ( 12 ). Therefore, we used the MEC cell line 1C6 to investigate a potential link between Aire expression and apoptosis.
To assess whether Aire overexpression could directly inhibit MEC division, we transfected 1C6 cells with vectors encoding either GFP or an Aire-GFP fusion protein and analyzed proliferation by BrdU incorporation. Comparable transfection effi ciencies were observed in the two cultures ( ‫ف‬ 30% GFP ϩ ). Correct folding, localization, and activity of the fusion protein was confi rmed by fl uorescence microscopy, Western blotting, and gene array analysis of transcription by cells transfected with Aire-GFP, with up-regulation of many PTAs (unpublished data). The proportion of GFP ϩ cells of immature epithelial cells in other tissues ( 11 ). To determine whether adult MEC lo were indeed precursors of Aire ϩ MECs, MEC lo were purifi ed and reaggregated with MHCmismatched embryonic stroma and adult CD4/8 singlepositive (SP) thymocytes. After 5 d of culture, a substantial proportion and number of MEC lo had up-regulated MHC II and Aire ( Fig. 3 D ). Aire ϩ MECs did not incorporate BrdU in vitro ( Fig. 3 D ), indicating that there was no expansion of trace Aire ϩ MEC lo and, therefore, that Aire Ϫ MEC lo were diff erentiating into the Aire ϩ cells observed. A proportion of these Aire + cells were MHC II lo , perhaps refl ecting slower kinetics of diff erentiation in vitro compared with in vivo and/or diff erential provision of the signals driving Aire and MHC II molecule up-regulation. Collectively, the observations that Aire ϩ MEC hi are postmitotic and are derived from cycling, Aire Ϫ MEC lo precursors establish that Aire expression is a late event in MEC diff erentiation.

Rapid turnover of Aire ؉ MECs
To investigate the fate of Aire ϩ MEC hi , we analyzed their longevity in BrdU decay experiments. Mice were exposed to BrdU for 2 wk to achieve maximal incorporation (80 -100%); label was then withdrawn and retention was measured at various times thereafter. During the chase period, BrdU ϩ TECs diminished in both label intensity and proportion labeled, suggesting further division by cycling cells and loss of cells by apoptosis or some other means ( Fig. 4 ).
Continued cycling, manifest as reduced BrdU intensity, was evident for the Aire Ϫ MEC lo and MEC hi subsets (unpublished data). The presence of a relatively large proportion of MEC lo with high levels of BrdU 4 wk after label withdrawal (unpublished data) indicates a low level of turnover and is in accord with low levels of division.
The label in the noncycling Aire ϩ MEC hi subset showed a rapid decline, with 90% of labeled cells gone 2 wk after withdrawal of BrdU ( Fig. 4 ). The rate of loss during the fi rst 2 wk (6.5% per day) is likely to be an underestimate (compared with the 13% per day observed in the incorporation study) because of the continued entry of labeled precursors during this time. Nevertheless, these data indicate very rapid turnover of this population relative to the Aire Ϫ MEC hi and MEC lo subpopulations. incorporating BrdU was not substantially diff erent between 2 and 3 d after transfection, indicating that Aire-GFP did not aff ect proliferation ( Fig. 5 C ). However, both the proportion and number of Aire-GFP ϩ MECs were decreased compared with GFP ϩ MECs from 3 d after transfection, declining until no Aire-GFP ϩ cells were detected after 5 d, whereas about one half of the GFP ϩ MECs remained ( Fig. 5 D ). Importantly, the specifi c loss of Aire-GFP -expressing MECs was caused by increased apoptosis at all time points ( Fig. 5 E ). Similar results were obtained with an Aire-GFP construct containing an internal ribosomal entry site before the GFP sequence, indicating that apoptosis was caused specifi cally by Aire (unpublished data). These data establish that overexpression of Aire does not directly inhibit MEC division but rather induces apoptosis. This fi nding off ers a likely explanation for the paucity of MEC lines that express Aire.

Conclusions
This study revealed several important aspects of TEC diff erentiation and population dynamics. A surprisingly high rate of turnover for the entire TEC population of 10% per day ( Ϯ 0.7%) was in accordance with previous estimates ( 7 ) and was driven by proliferation, predominantly within the Aire Ϫ MEC hi subset. The rapid cycling of all cells in this subset strongly suggests that it constitutes an MEC transit-amplifying population that may regulate parameters such as overall numbers of MECs and the tempo of their diff erentiation. The fi nding that certain TECs are highly proliferative also raises questions regarding the long-held assumption that TECs are relatively radioresistant because of low mitotic activity: does radiation in fact cause loss of TEC subsets and subsequent defects in thymocyte diff erentiation and tolerance?
Direct analysis of BrdU incorporation by Aire ϩ MECs at the single-cell level revealed that, in the adult, these cells are postmitotic and derive from a cycling precursor. While this paper was under review, Rossi et al. ( 13 ) showed maturation of E15 CD80 Ϫ to CD80 ϩ MECs but also that CD80 was not a surrogate marker for Aire expression. Transcription of Aire in the ex vivo CD80 Ϫ MEC subset left open the question of the precise identity of Aire ϩ MEC precursors in the fetal thymus. High levels of CD80 and MHC II were better correlates for Aire expression in adult MECs, and reaggregate thymus organ culture of purifi ed MEC lo gave rise to Aire ϩ MEC hi , without and -APC. Suspensions of thymic stromal cells were prepared as previously described ( 17 ). All digests were counted and analyzed for TEC enumeration, and totals were compared using the Student ' s t test. Intracellular Aire staining was performed using fi xation/permeabilization buff ers (eBioscience) according to the manufacturer ' s instructions. For BrdU experiments, the second of two enzymatic digestions of individual thymi was used for analysis. Stromal cells were fi xed/permeabilized using BD Bioscience buff ers, followed by paraformaldehyde/Tween 20, and were treated with DNase (50 Kunitz units) before staining with anti-BrdU. Data were acquired on a fl ow cytometer (LSR II; BD Biosciences) and analyzed using FloJo software (TreeStar, Inc.).
Reaggregate thymus organ cultures. MEC lo and SP thymocytes from B6 mice were sorted to Ͼ 97% purity on a cytometer (MoFlo; DakoCytomation), as previously described ( 7 ). MEC lo and SP thymocytes were reaggregated with suspensions of fresh E15.5 embryonic stroma from B6.H-2 g7 mice, as previously described ( 13 ), at ratios of 1:1:8. Reaggregates were dispersed in dispase/DNase for fl ow cytometric analysis. For analysis of proliferation, 10 M BrdU was added 5 h before termination of the culture.
Immunohistology. 8-m air-dried cryosections of thymus were stained with primary antibodies for 30 min and then subjected to three 5-min washes in PBS. Secondary antibodies were incubated for 30 min, followed by washes. Sections were then incubated in DAPI (Invitrogen) for 5 min, washed, and mounted with coverslips in aqueous mounting medium (Biomeda). Images were acquired with a confocal microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) using a xenon-arc lamp in a wavelength switcher (Lambda DG-4; Sutter Instrument Co.) and processed with Slidebook imaging software (Intelligent Imaging Inc.).
Cell lines and transfection. The 1C6 cell line, originally derived from mouse MECs ( 18 ), was a gift of M. Kasai (National Institute of Infectious Diseases, Tokyo, Japan). Cells were seeded onto 12-well plates (at 10 -20% confl uence) and transfected with Eff ectene (QIAGEN) and 300 ng of either aire cloned into pEGFP-c1 expression vectors (Aire-GFP) or 300 ng of pEGFP-c1 (GFP) 16 h later. Transfected 1C6 cells were harvested at a various time points, and the relative intensity of GFP was analyzed by fl ow cytometry. Apoptosis was measured with Annexin V and DAPI staining, and GFP ϩ MECs were analyzed by fl ow cytometry. expansion of low-level ( ‫ف‬ 1%) Aire ϩ MEC lo . The low efficiency of MEC lo transition to Aire ϩ MEC suggests that only a minor subpopulation of MEC lo exhibits this potential. The infl uence of apoptosis on the emerging Aire ϩ population does need to be taken into account, however, as does the possibility that not all MEC lo are exposed to the necessary inductive signals in this system. Whether the diff erentiation of Aire Ϫ MEC lo to Aire ϩ MEC hi occurs via an Aire Ϫ MEC hi intermediate awaits the means to purify this latter population. Furthermore, the dependency of this adult diff erentiation program upon signals important for fetal Aire ϩ MECs (i.e., the requirement of RANK signals) ( 13 ) remains to be determined.
The Aire ϩ MEC hi population has a high turnover, with loss of cells most likely through apoptosis, based on the in vivo kinetics of decay and in vitro overexpression studies. The possibility that some in vivo loss of BrdU ϩ Aire ϩ MEC hi occurs by cells down-regulating Aire and joining another subset cannot be ruled out. Nevertheless, the fi ndings of this study are consistent with the terminal diff erentiation model and not the progressive restriction model of TEC diff erentiation.
The induction of apoptosis by Aire further supports the fi nality of its activity in MEC diff erentiation. Interestingly, the highest levels of apoptosis were seen 4 d after transfection, suggesting that Aire may initiate a sequence of events that culminates in MEC death. This scenario raises the question of whether apoptosis occurs via direct induction of an apoptotic program or indirectly through up-regulation of PTA that overload the protein synthesis machinery to such an extent as to provoke death through ER stress. Regardless, the delay in apoptosis should allow suffi cient time for the promiscuous expression of genes by MECs, followed by cell death to deliver a battery of PTAs to dendritic cells for crosspresentation and induction of central tolerance to peripheral self ( 14 ). Thus, to aff ect central tolerance, Aire may not only induce transcription of PTA but also promote cellular changes that ensure their effi cient presentation.

Mice.
Aire-defi cient mice were derived and genotyped as previously described ( 2 ) and were analyzed on the C57BL/6 (B6) ϫ 129 genetic background. Mice were housed at the Center for Animal Resources and Comparative Medicine at the Harvard Medical School, and the Institutional Animal Care and Use Committee approved procedures. BrdU labeling experiments were initially performed with 6 -8-wk-old NOD ϫ B6 mice, then with B6 mice for fi nal replicates. B6.H-2 g7 mice ( 15 ) were time mated, and the day of vaginal plug detection was designated E0.5.
BrdU administration. BrdU was administered as previously described ( 16 ). In brief, mice were injected intraperitoneally with 1 mg BrdU (Sigma-Aldrich) in PBS to provide a labeling pulse. Continuous labeling was maintained by incorporation of 0.8 mg/ml BrdU in sterile drinking water with artifi cial sweetener. 8-wk-old mice were used for this experiment because thymic stromal cell numbers remain essentially at steady state during the labeling period ( 7 ). Fluorescent reagents and fl ow cytometry. The following antibodies, purchased from BD Biosciences unless otherwise stated, were used: rabbit antimouse K5 (Covance), rat anti-Aire (rat IgG2c, clone 5H12; a gift from H. Scott, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia),