Foxn1 Expression in the Developing, Aging, and Regenerating Thymus Immanuel Rode, Vera C. Martins, Günter Küblbeck, Nicole Maltry, Claudia Tessmer and Hans-Reimer Rodewald This information is current as of September 25, 2021. J Immunol 2015; 195:5678-5687; Prepublished online 4 November 2015; doi: 10.4049/jimmunol.1502010 http://www.jimmunol.org/content/195/12/5678 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Foxn1 Protein Expression in the Developing, Aging, and Regenerating Thymus

Immanuel Rode,* Vera C. Martins,*,1 Gunter€ Kublbeck,*€ Nicole Maltry,* Claudia Tessmer,† and Hans-Reimer Rodewald*

The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expres- sion patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon chal- lenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the Downloaded from responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1+Ly51+CD802 TECs, castration led to expansion of Foxn1+Ly512CD80+ TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1low or Foxn12 TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations. The Journal of Immunology, 2015, 195: 5678–5687.

nactivating mutations in the forkhead box N1 (Foxn1) stage in a cell-intrinsic manner (1, 2, 9–13). As a consequence, the http://www.jimmunol.org/ cause the athymic “nude” phenotype in humans, rats, and Foxn1-deficient epithelium remains alymphoid and TECs fail to I mice (1, 2). The 10 Foxn1 exons encode a 648-aa tran- further differentiate into cortical and medullary subsets. scription factor of the forkhead box family. The first noncoding The discovery of Foxn1 as the gene mutated in nude mice dates exon 1 is alternatively transcribed, and Foxn1 mRNA comes in back .20 y (1, 3), yet until now it has been virtually impossible to two isoforms as either exons 1a–9 or exons 1b–9 (3). Within the study this important protein. For example, Foxn1 protein expres- Foxn1 protein, the evolutionary conserved DNA binding domain sion has not been amenable to flow cytometry on TEC subsets or and the activation domain have been functionally characterized to immunoprecipitation. Levels of Foxn1 expression have mostly in vitro (4, 5). been inferred from analysis of RNA that suggested declining The thymus, the main site of T cell development, arises from a Foxn1 expression to parallel age-dependent thymus involution (4, by guest on September 25, 2021 shared parathyroid/thymus anlage from third pharyngeal pouch en- 5, 14). Introduction of Foxn1 alone appears to be sufficient to doderm. Separation of the common anlage coincides with the arrival reprogram mouse embryonic fibroblasts into functional TECs (6, of hematopoietic precursor cells from the bone marrow inside the 7, 15), suggesting that Foxn1 is a determining master regulator of thymus at embryonic day (E)12 (6, 7). Although Foxn1-deficient TEC development even beginning from non-TECs. mutants lack a functional thymus, a Foxn1-deficient thymic anlage In this study, we report the generation and application of new nevertheless forms and migrates to its final mediastinal position robust tools to study Foxn1 protein expression. We analyzed thymic above the heart (8). Lack of Foxn1 function causes a developmental Foxn1 expression throughout most of the embryonic and adult life arrest in thymic epithelial cells (TECs), presumably at an immature of a mouse. Additionally, we investigated Foxn1 expression in the absence of normal T cell development, as well as in the course of thymic insults and regeneration. The results show that already in *Division of Cellular Immunology, German Cancer Research Center, D-69120 Hei- young mice large fractions of bona fide TECs are Foxn12/low, delberg, Germany; and †Genomics and Proteomics Core Facility, Monoclonal Anti- body Facility, German Cancer Research Center, D-69120 Heidelberg, Germany precluding Foxn1 as a universal TEC marker, and that Foxn1 is a 1Current address: Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal. highly dynamic protein in response to thymic injury. ORCID: 0000-0001-8717-9709 (V.C.M.). Received for publication September 9, 2015. Accepted for publication October 13, Materials and Methods 2015. Mice and targeting

This work was supported in part by Deutsche Forschungsgemeinschaft–Sonderfor- lacZ/lacZ 2/2 schungsbereich Grant 873-Project B11, as well as by a grant from the Helmholtz Foxn1 (8), Foxn1-Cre (16), Rosa26-YFP (17), and Rag2 (18) Alliance Preclinical Comprehensive Cancer Center (to H.-R.R.). mice were described earlier. C57BL/6J mice were either bred at the animal facility of the German Cancer Research Center or purchased from Charles Address correspondence and reprint requests to Dr. Immanuel Rode and Dr. Hans- River Laboratories. Reimer Rodewald, Division of Cellular Immunology, German Cancer Research Cen- HA/HA ter, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail addresses: Foxn1 knock-in mice were generated by conventional gene tar- [email protected] (I.R.) and [email protected] (H.-R.R.) geting using the construct shown in Supplemental Fig. 1A. All animal experiments were performed in accordance with institutional and gov- The online version of this article contains supplemental material. ernment regulations and were approved by the Regierungspra¨sidium Abbreviations used in this article: cTEC, cortical TEC; DEX, dexamethasone; (Karlsruhe, Germany). E, embryonic day; FDG, fluorescein-di-b-D-galactopyranoside; Foxn1, forkhead box N1; HA, hemagglutinin; mTEC, medullary TEC; rhFGF7, recombinant human fibro- Epithelial cell isolation blast growth factor 7; TEC, thymic epithelial cell. Thymic stromal cell isolation was done with slight modifications as de- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 scribed earlier (19). In contrast to the published method, minced thymi www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502010 The Journal of Immunology 5679 were directly subjected to digestion without discarding any fractions, and Expression systems lymphoid cells (anti-CD45 beads, Miltenyi Biotec) and erythrocytes (anti- Ter119 beads, Miltenyi Biotec) were manually depleted using MACS Foxn1 and fragments of various lengths were cloned into pEGFP-C1 and technology. Embryonic thymi were digested in 96-well V-bottom plates, electroporated (Gene Pulser Xcell, Bio-Rad) into Cos-1 cells for transient and the resulting single-cell suspensions were directly used for flow expression. cytometry without depletion. For immunization, Foxn1 cDNA was cloned into pET-22b(+) (Nova- Alveolar epithelial cells were isolated from the lungs as described (20). gen). Expression of the protein in BL21-CodonPlus (DE3)–RIL bacteria b Small intestinal epithelial cells were isolated by incubating cleaned and (Agilent Technologies) was induced with isopropyl -D-thiogalactopyranoside finely minced small intestine fragments three times for 20 min each at (Sigma-Aldrich). 37˚C in RPMI 1640 (5% FCS, 1.25 mM EDTA) while shaking. Super- X-gal staining natants were filtered (70 and 30 mm) to yield single-cell supensions. Newborn thymi were fixed for 15 min with 2% paraformaldehyde on ice, Flow cytometry and counting transferred into X-gal staining solution, and incubated at 37˚C with gentle For intracellular stainings (Foxp3 staining kit, eBioscience), cells were first agitation. The staining reaction was stopped by repeated washes with un- incubated with a Live/Dead fixable violet dead cell stain kit (Life Tech- supplemented PBS. Staining solution (in unsupplemented PBS) included nologies) and then Fc blocked, and surface stainings were directly blocked. 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, Simultaneous staining of YFP and Foxn1 required replacing the eBio- 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 1 mg/ml X-gal. science fixative with 3.7% paraformaldehyde in PBS. Fluorescein-di-b-D- galactopyranoside (FDG) staining was done with the FluoReporter lacZ flow cytometry kit (Life Technologies). Abs used were CD45 (30F11) Results Brilliant Violet 421/PE-Cy7, Aire (5H12) Alexa Fluor 488, Ki67 (SolA15) New tools to measure Foxn1 expression at the single-cell level PE-Cy7 (eBioscience), CD326 (G8.8) PerCp-Cy5.5/PE-Cy7/Alexa Fluor 647, CD80 (16-10A1) Brilliant Violet 605, rat IgG1, k (RTK2071) PE-Cy7 Most information on Foxn1 expression is based on RNA data or Downloaded from (BioLegend), Ly51 (6C3) PE, CD45 (30F11) FITC (BD Biosciences), anti- knock-in/transgenic reporter alleles (8, 14, 21). However, Foxn1 hemagglutinin (HA; GG8-1F3.3.1) PE (Miltenyi Biotec), and Foxn1 (2/41) protein and reporter levels may differ due to transcriptional vari- Alexa Fluor 647 (available on request). ations, posttranslational modifications, or differences in protein For TEC counting, Accudrop beads (BD Biosciences) of a fixed con- stability comparing the reporter and the native Foxn1 protein. To centration were spiked into all samples just before they were acquired on the LSRFortessa. overcome these disadvantages, we followed two different experi-

mental approaches. First, we engineered a mouse carrying a tag- http://www.jimmunol.org/ Ab generation and labeling ged version of Foxn1, and second, we generated a monoclonal, Full-length, native, recombinant mouse Foxn1 was used to immunize mice flow cytometry grade Ab. Because modifications of the Foxn1 in collaboration with the mAb Facility at the German Cancer Research locus had previously resulted in hypomorphic alleles (22), we Center. One purified clone (2/41, mouse IgG2b) was labeled for all further decided to attach the small HA tag of 9 aa to the C terminus of experiments using the Alexa Fluor 647 Ab labeling kit (Life Technologies). Foxn1 by gene knock-in in embryonic stem cells (Supplemental Dexamethasone treatment Fig. 1A, 1B). Homozygous Foxn1HA/HA mice were born with fur Seven- to eight-week-old Foxn1HA/HA mice received a single i.p. injection and thymus, indicating that the mutant allele encodes a functional of either 20 mg/kg dexamethasone (DEX; Sigma-Aldrich) dissolved in Foxn1 protein. We compared total thymus cell numbers and DMSO (Sigma-Aldrich) or an equal volume of DMSO. phenotypic stages of T cell development in Foxn1 HA/HA to Foxn1+/+ by guest on September 25, 2021 Recombinant human fibroblast growth factor 7 injection C57BL/6 mice (Supplemental Fig. 1C–E). All parameters were comparable, except for an increase in cell numbers at 6 wk of age in Six-day-old C57BL/6J mice were i.p. injected with either 5 mg/kg recom- early backcrosses of Foxn1 HA/HA mice compared with C57BL/6 binant human fibroblast growth factor 7 (rhFGF7; Kepivance [palifermin] controls (Supplemental Fig. 1C). Reanalysis of 10th generation from Sobi) or an equal amount of solvent (PBS) for 3 consecutive days. HA/HA Three (i.e., postnatal day 9) and six days later (i.e., postnatal day 12), mice backcrosses of Foxn1 mice revealed comparable cell numbers were analyzed by flow cytometry. also in 6- to 8-wk-old mice (Supplemental Fig. 1C, 1E, red sym- bols). We next analyzed whether the HA-tagged Foxn1 protein Surgical castration showed a nuclear localization in TECs. Double staining against For surgical castration, 9- to 11-wk-old C57BL/6J mice were anesthetized Foxn1-HA and the epithelial cell–specific and a small ventral abdominal midline incision was made to reveal the DNp63 revealed colocalization within the nuclei of TECs (Fig. testes. These were removed along with surrounding fatty tissue. Sham castration was performed using the same surgical procedure, but without 1A). Collectively, these data show that HA-tagged Foxn1 correctly removal of the testes. localizes to the nucleus and functions well in lieu of the normal protein. Histology In the second approach, we immunized mice with full-length Embryonic day 14.5 embryos, used for thymus staining in Fig. 1A, were recombinant native Foxn1 to produce mouse mAbs. One mAb fixed overnight (4% paraformaldehyde, 4˚C), and 1-wk-old thymus lobes (clone 2/41) was selected for detailed characterization. This Ab (Supplemental Fig. 2B) from C57BL/6J and Foxn1HA/HA mice were fixed for 1.5 h. Fixation was followed by cryoprotection in 20% sucrose/PBS (w/v). stained Cos-1 cells transiently transfected with Foxn1 cDNA After OCT embedding in 280˚C isopentane, embryonic sections were (Supplemental Fig. 2A). As further evidence for the specificity stained with anti-DNp63 (Poly6190, BioLegend) and anti-HA (3F10, Roche). of both reagents, double staining with anti-Foxn1 and anti-HA Anti-DNp63 was detected with anti-rabbit Alexa Fluor 488 (A-11008, Abs identified the same cells in Foxn1 HA/HA thymus sections Thermo Fisher Scientific), and anti-HA was detected with anti-rat Alexa (Supplemental Fig. 2B). Epitope mapping with deletion constructs Fluor 546 (A-11081, Thermo Fisher Scientific). Endogenous biotin in thymus sections was blocked (E-21390, Thermo Fisher Scientific) before incubation determined that clone 2/41 binds the C terminus of Foxn1 between with anti–Foxn1Bio (2/41) and anti-HA (3F10, Roche). The biotinylated amino acids 475 and 542 (Supplemental Fig. 2C). (EZ-Link-kit 21327, Thermo Fisher Scientific) Foxn1 Ab was detected with Next, we tested the capacity of Abs against HA or Foxn1 (clone anti–biotin-Cy3 (Jackson ImmunoResearch Laboratories), and anti-HA was 2/41) to recognize the Foxn1-HA fusion protein or normal Foxn1 detected with anti-rat Alexa Fluor 488 (A-11006, Thermo Fisher Scientific). protein, respectively, by flow cytometry. Newborn TECs from Southern blotting Foxn1+/+ or Foxn1HA/HA mice were intracellularly stained with Nonradioactive Southern blotting was performed on SspI/SpeI-digested Abs against HA, the new Foxn1 Ab (clone 2/41), or a combination tail DNA of Foxn1HA/HA knock-in mice using a biotinylated Foxn1 probe of both, and analyzed by flow cytometry. Whereas the anti-HA Ab (Supplemental Fig. 1A). stained the Foxn1HA/HA TECs exclusively (Fig. 1B, left column), 5680 Foxn1 PROTEIN IN SPACE AND TIME

FIGURE 1. New Foxn1 tools are TEC spe- cific. (A) E 14.5 thymus sections of either Downloaded from Foxn1+/+ (upper panel)orFoxn1HA/HA (lower panel) mice were stained with Abs against HA (red, left) and DNp63 (green, middle) and counterstained with DAPI (blue, merge on the right; scale bars, 20 mm). One representative out of two independent experiments is shown. (B) 2

Newborn thymic stroma (live, CD45 ) of either http://www.jimmunol.org/ Foxn1+/+ (upper panel)orFoxn1HA/HA mice (lower panel) was intracellularly stained with Abs against HA (left), Foxn1 (middle), or a combination of both (right). Note that the epi- tope of the Foxn1 Ab is not affected by the presence of the C-terminal HA tag (middle). One representative out of three experiments is shown. (C) Newborn thymic stroma (live, CD452)of Foxn1+/+ (left), Foxn1+/lacZ (middle), or homo- zygous nude Foxn1lacZ/lacZ mice (right) was by guest on September 25, 2021 stained with either FDG (upper panel) or anti- Foxn1 (lower panel). Note that the live, CD452 EpCAM2 population shows no signs of Foxn1 staining. One representative out of three inde- pendent experiments is shown.

the tag-independent anti-Foxn1 Ab stained TECs of both geno- promoter (8). According to their allelic composition, TECs of this types (Fig. 1B, middle column). When both Abs were used in strain expressed homozygously Foxn1 (Foxn1 +/+)orhomozy- combination, Foxn1HA/HA but not Foxn1+/+ TECs were double gously b-galactosidase (Foxn1 LacZ/LacZ)orbothheterozygously positive, that is, both Abs stained the same cells (Fig. 1B, right (Foxn1LacZ/+), which is consistent with the blue X-gal staining of column). To further exclude nonspecific labeling, we used Foxn1 LacZ whole thymi from the different genotypes (Supplemental Fig. knock-in mice, in which Foxn1 is nonfunctional whereas b-galacto- 2D). Replacing chromogenic X-gal with flow cytometry suitable sidase is produced under the control of the endogenous Foxn1 FDG allowed us to analyze TECs from these genotypes by flow The Journal of Immunology 5681 cytometry. Simultaneous Foxn1 and FDG staining of cells was Foxn1 expression during ontogeny technically incompatible, and hence we stained cells either by LacZ Specification of pharyngeal epithelium toward TECs occurs in- FDG (indicating at least one Foxn1 allele) (Fig. 1C, top row) dependent of Foxn1, but further differentiation into cortical and Foxn1 + or with anti-Foxn1 Ab (indicating at least one allele) medullary TECs requires Foxn1 (8). During ontogeny, compre- (Fig. 1C, bottom row) versus the pan-epithelial marker EpCAM. hensive information on Foxn1 expression levels and the distribu- Wild-type Foxn1 +/+ TECs stained for Foxn1 only (Fig. 1C, left tion of expressing TEC subsets is lacking. Analysis of embryonic column), heterozygous Foxn1 +/LacZ TECs were positive for FDG development (E14.5–18.5) revealed that ∼94% of CD452EpCAM+ and anti-Foxn1 (Fig. 1C, center column), and homozygous nude Foxn1 LacZ/LacZ TECs were exclusively positive for FDG (Fig. 1C, epithelial cells in the thymus expressed Foxn1 (Fig. 2A). Non- epithelial (CD452EpCAM2)stromalcellswereFoxn12 (Fig. 2A). right column). Therefore, cells with “TEC identity” could be + identified in the Foxn1-deficient thymic rudiment by the active These high percentages of Foxn1 TECs remained constant over time in prenatal development (Fig. 2B, 2C), irrespective of TEC Foxn1 promoter (evident by staining with FDG), but these 2 “wannabe” TECs cannot express Foxn1, and lack of their staining subset and proliferative state. The small fraction of Foxn1 TECs 2 2 proves the specificity of the monoclonal Foxn1 Ab (clone 2/41). In was initially mostly limited to Ly51 CD80 cells (termed double- + 2 line with this specificity, the CD452EpCAM2 non-TEC stroma negative TECs in this study) and to Ly51 CD80 cells (termed 2 remained unstained (Fig. 1C, bottom row). Moreover, we also Ly51+ cortical TECS [cTECs]), but some Ly51 CD80+ cells stained nonthymic epithelium and found that the tested alveolar (termed CD80+ medullary TECs [mTECs]) were also detected (Supplemental Fig. 2E) and intestinal (Supplemental Fig. 2F) (Fig. 2A). 2 epithelial cells were not recognized by the Foxn1 Ab based on To determine whether Foxn1 cells (Fig. 2C) represent a pre- or Downloaded from flow cytometry. post-Foxn1 stage, we lineage-traced these cells using Foxn1::Cre http://www.jimmunol.org/

FIGURE 2. Foxn1 expression remains unal- tered from E14.5–18.5. (A) Single thymi of developing C57BL/6J mouse embryos were by guest on September 25, 2021 enzymatically digested and analyzed for Foxn1 expression after gating on live, CD452 events. Foxn1 expression by thymic stromal cells at E14.5 (upper panel) and E18.5 (lower panel)is shown on the left. The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. Expression of Ly51 versus CD80 on Foxn12 (middle) and Foxn1+ (right) TECs (live, CD452, EpCAM+) is shown. One representative experiment out of two is shown (n = 5 per age and experiment). (B) Frequency of Foxn1+ TECs (live, CD452, EpCAM+) in individual embryonic thymi from E14.5 to E18.5 (n = 10 per age, mean 6 SD). (C) Frequency of Foxn12 TECs (live, CD452, EpCAM+) in individual thymi from E14.5 to E18.5 (n = 10 per age, mean 6 SD). (D) Lineage tracing using Rosa26YFP (left)or Foxn::Cre x Rosa26YFP transgenic littermates (right) at E14.5. Expression of YFP and its correlation with Foxn1 expression by TECs (live, CD452, EpCAM+) is shown. One repre- sentative experiment out of two is shown. 5682 Foxn1 PROTEIN IN SPACE AND TIME transgenic mice crossed to Rosa26-YFP reporter mice (16, 17). In Finally, the injection of Kepivance (palifermin), a DN23KGF form these mice all cells expressing Foxn1 at any time point in their of rhFGF7, has been shown to have regenerating capacity on the development and their progeny are YFP+. In E14.5 thymi from thymus (33–35). Foxn1::Cre transgenic mice, but not Cre2 control mice, almost all DEX injection affected female and male Foxn1HA/HA mice alike TECs had a history of Foxn1 expression (Fig. 2D). The vast ma- by reducing thymocyte and TEC numbers drastically within 2 d. jority of these cells actively expressed Foxn1 (YFP+Foxn1+). By day 7, there was a clear, albeit not yet complete, recovery of However, ∼5% of these lineage-traced cells were Foxn1 protein- cellularity (Fig. 4A, 4B). After 2 d, Foxn1 expression dropped negative (YFP+Foxn12) (Fig. 2D). Collectively, during embryonic markedly in response to DEX treatment (Fig. 4C, 4D). In addition development, TECs almost homogeneously express Foxn1, and to its global TEC diminishing effect, DEX strongly influenced consequently this is true for TECs with cTEC and mTEC phe- the ratio of Foxn1+CD80+ mTECs to Foxn1+Ly51+ cTECs notypes. A very small fraction of cells (∼5%) has gone through a (Supplemental Fig. 3A). The reduction in Foxn1 expression was Foxn1 RNA-expressing stage, but fails to express the protein. temporary, that is, 7 d after injection the frequency of Foxn1- Hence, these cells are “post-Foxn1 mRNA” cells. Additionally, a expressing cells had already returned to normal (Fig. 4C, 4D). minor subset of EpCAM+YFP2Foxn12 cells was also found. Regrowth of Ly512CD80+Foxn1+ TECs did not occur within the 7-d observation period, and hence the altered ratio of Foxn1+ Foxn1 expression during adulthood CD80+ mTECs to Foxn1+Ly51+ cTECs persisted (Supplemental We examined Foxn1 expression in Foxn1HA/HA mice using our Fig. 3B). In summary, DEX treatment has a profound effect on Foxn1 mAB 2/41 between 11 d after birth and up to ∼2 y of age. Foxn1 expression in the thymus, on proportions of Foxn1-expressing

Flow cytometric analysis using anti-Foxn1 revealed that both TEC subsets and on their cycling properties. Whereas Foxn1 ex- Downloaded from expression levels and frequencies were highest in the youngest pression recovers within 7 d, TEC homeostasis does not appear to analyzed mice (Fig. 3A–C), which was verified with an inde- be equilibrated by this time point. pendent dataset obtained by anti-HA staining. Frequencies of Next, we stimulated mice with rhFGF7. rhFGF7 has been shown Foxn1-expressing cells progressively declined with time (Fig. 3A, to have a direct effect on FgfR2-IIIb+ TECs and to cause an initial 3B). Interestingly, this decline began within the first weeks after thymocyte loss followed by a drastic increase in thymic cellularity 2 birth (Figs. 2A, 3A, 3B) and reached a plateau of ∼50% Foxn1 (33–35). As a result, the stimulated thymus increases in size, in http://www.jimmunol.org/ TECs by 10 wk (Fig. 3A, 3B), which remained constant throughout contrast to the recovery to normal size following DEX treatment the observation period. On a per-cell basis, however, Foxn1 (28). Injection of rhFGF7 triggered an initial reduction of thy- downregulation continued throughout life (Fig. 3C). Based on mocyte numbers (Fig. 5A) and, already by day 3, an increase in expression of the markers Ly51 and CD80, cells marked by TEC numbers (Fig. 5B), which is consistent with stimulated TEC Foxn1 expression always included the major TEC populations proliferation on day 3 (Fig. 5C). The frequency of Foxn1- (Fig. 3D). In contrast, among the Foxn12 cells, double-negative expressing cells declined transiently on day 3 (Fig. 5D). Akin to TECs dominated (Fig. 3E). Taken together, we observed a the DEX recovery, the ratio of Foxn1+CD80+ mTECs to Foxn1+ temporal loss of Foxn1-expressing cells together with a long- Ly51+ cTECs was altered (Supplemental Fig. 4A). Among lasting, yet slow downregulation of Foxn1 levels in the remaining Foxn12 and Foxn1+ TECs, Ly51+ cTECs expanded (Supplemental by guest on September 25, 2021 expressers. Fig. 4A). Hence, this early thymic stimulation was accompanied To examine whether Foxn1 expression levels are dependent by subset changes remarkably similar to the ones observed late on normally proceeding T cell development, we analyzed Foxn1 during DEX regeneration. The initially major Ly512CD80+ expression in Rag2-deficient mice. In these mutants, T cell de- Foxn1+ compartment of control mice was replaced by Ly51int velopment is blocked at the double-negative 3 stage. This state is CD802Foxn1int TECs, which turned into a Ly51+CD802Foxn1hi associated with impaired TEC differentiation, which can be population by day 6 (Fig. 5E, right column, Supplemental Fig. corrected by reconstitution with wild-type thymocytes (23). In- 4A). Note that the level of Foxn1 expression on these TECs triguingly, in the presence of only immature thymocytes (double- exceeded the level of control TECs (Fig. 5E, left column). negative 3), a very high fraction of TECs expressed Foxn1 up to In our final model of thymus modulation, we castrated mice and 28 wk (Fig. 3F, 3G). Hence, progression beyond the double- followed Foxn1 expression over time. Castration led to an increase negative 3 stage induces loss or downregulation of Foxn1, possi- in thymocyte numbers as early as 8 d after surgery (Fig. 6A). bly by inducing TEC maturation. Frequencies of Foxn1 expression did not show major alterations (Fig. 6B). On a per-cell basis, however, all Foxn1-expressing cells Foxn1 during thymic regeneration had shifted toward a higher expression level at day 14 posttreat- Loss of Foxn1 during adulthood has been linked to thymic invo- ment, similar to that of young mice (Fig. 6C). This effect was lution (14), an assumption that is supported by the premature observed as early as day 8, when thymocyte numbers were already thymic degeneration caused by genetic reduction of Foxn1 ex- increased, in one third of the cases, and in 100% of the animals at pression (22, 24). Corroborating these findings, elevated Foxn1 day 14 after surgery, when thymocyte numbers were even higher. expression has been shown to attenuate thymic involution in Therefore, upregulation of Foxn1 does not seem to be an absolute multiple settings (24–26). Hence, we were prompted to elucidate prerequisite for higher thymopoietic capacity at day 8 of recovery. Foxn1 expression at the single-cell level in response to thymic In marked contrast to the treatments described above, where insults and/or thymic regeneration. Three conditions are com- thymic regeneration led to the appearance of a Ly51+Foxn1+ monly induced in mice to study thymus regeneration, that is, population (Fig. 5E, Supplemental Fig. 3B), CD80+Foxn1+ TECs glucocorticoid treatment, growth factor treatment, or castration. represented the most abundant population in response to castration Glucocorticoids such as DEX are well known for their multiple (Fig. 6D). This shift in TEC distribution could be first observed detrimental effects on both thymocytes and TECs. In contrast to 8 d after surgery, and became more prominent 14 d after treatment thymic aging, the DEX effect is transient, and thymocyte as well when these cells uniformly turned Foxn1hi (Fig. 6D, Supplemental as TEC numbers recover (27, 28). Conversely, thymus regenera- Fig. 4B). Collectively, compared with DEX and rhFGF7 treat- tion can be induced by castration of mice, which results in a ment, castration has a unique effect on the thymus by strongly dramatic, but transient, increase of thymus cellularity (23, 29–33). expanding CD80+ Foxn1hi TECs. The Journal of Immunology 5683 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Loss of Foxn1 expression with age depends on lymphocytes. (A) Flow cytometry for Foxn1 expression by thymic stroma (live, CD452) isolated at the indicated time points. The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. One representative out of three to four experiments is displayed. (B) Summary of Foxn1 expression by TECs. Frequencies of Foxn1+ (n) and Foxn12 (s) TECs (live, CD452, EpCAM+, n = 4–5 per age, mean 6 SD) are shown. (C) Histogram based on FACS plots shown in (A). Foxn1 stainings of the TEC compartment at various ages are overlaid to visualize differences in expression levels. Phenotype of Foxn1+ (D) and Foxn12 (E) TECs (live, CD452, EpCAM+) isolated from mice of various ages (as indicated) is shown. One representative out of three to four experiments is displayed. (F) Representative staining depicting Foxn1 expression by thymic stroma (live, CD452) derived from Rag2-deficient mice of various ages. The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. One representative out of three to four experiments is shown. (G) Summary of the frequencies of Foxn12 (s) and Foxn1+ (n) TECs from Rag2-deficient mice at various ages (n = 6–8 per age, mean 6 SD). 5684 Foxn1 PROTEIN IN SPACE AND TIME Downloaded from http://www.jimmunol.org/ FIGURE 4. TEC rebound following DEX treatment. Seven- to 8-wk-old Foxn1HA/HA mice were injected once with DMSO or 20 mg/kg DEX in DMSO and thymic regeneration was analyzed at 2 d (n, n = 6 per treatment) and 7 d (s, n = 9 per treatment) after injection. Females (red) and males (blue) were analyzed separately. Data represent three independent experiments with two to three mice per group (mean 6 SD). Absolute thymocyte (A) and TEC (B) numbers are shown. The frequency of Foxn1-expressing TECs is depicted in (C). (D) Stroma (live, CD452)-specific expression of Foxn1 at 2 (left)or7d after injection (right). The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. One representative out of three experiments is shown. Note that the alterations in EpCAM staining are a result of differently labeled (dye) EpCAM Abs.

2 Discussion addressed the origin of the few Foxn1 TECs in embryonic mice. by guest on September 25, 2021 Reliable detection of the Foxn1 protein has been a notorious hurdle In line with observations of others, most TECs displayed a history in studies on its roles in thymus organogenesis and thymus epi- of Foxn1 expression. Costaining of lineage-traced embryonic thelium. We generated a C-terminal–tagged Foxn1HA allele and a TECs with anti-Foxn1 Ab identified some EpCAM+ TECs that do flow-grade Foxn1 Ab. In contrast to a published hypomorphic not actively express Foxn1 and had not expressed it in the past. Foxn1 allele (22), Foxn1HA/HA mice have a phenotypically and The significance of this population is unclear, but it may be of functionally normal thymus. minor importance: due to their EpCAM expression, they cannot Although data with anti-Foxn1 serum have been reported (21, represent the recently proposed EpCAM2Foxn12 stem cells, and 36), commercially available antisera did not work robustly in our second, they are unable to rebuild the thymus after prenatal intox- hands to detect Foxn1. We therefore, in a second approach, raised ication of Foxn1+ TECs (38, 39). These cells may originate from a tag-independent mAb against endogenous Foxn1. We have the incomplete separation of the parathyroid and the thymus (40), mapped the epitope of this mAb to the activation domain, close to as has been proposed previously for the cervical thymus in a re- the C terminus of Foxn1. The Ab detects Foxn1 by immuno- ciprocal manner (21). fluorescence, by flow cytometry, and by immunoprecipitation in Whereas the embryonic and young thymi contain only a small thymus cell lysates. 2 2 HA/HA fraction of Foxn1 cells, the proportion of Foxn1 cells increases Foxn1 mice and the Foxn1-specific mAb enabled us to between 2 and 10 wk, reaching a plateau of roughly 50% Foxn12 assess Foxn1 protein expression during thymus development, in 2 TECs. Based on their Foxn1 expression history, these Foxn1 TECs thymi with arrested T cell development, during thymus aging, and 2 are post-Foxn1 cells rather than representing a Foxn1 lineage after thymic insult, as well as regeneration. During prenatal de- (37, 38). Although the Foxn1+ TECs contain all known mature velopment and up until about 2 wk of age, a very high percentage major TEC subsets as well as few double-negative TECs, the latter of all TECs were Foxn1+. By 4 wk of age, still .70% of TECs fraction dominates the Foxn12 TEC population. Currently, we expressed Foxn1. An earlier report using rabbit anti-Foxn1 serum + 2 quantified numbers of Foxn1+ TECs on cytospins and obtained lack information on the relative location of Foxn1 and Foxn1 TEC subsets in the thymus. Thus, neither the location nor the much lower frequencies (36). The reason for this discrepancy is 2 unclear, but it is possible that the representation of Foxn1+ TEC function of Foxn1 TECs is known. We speculate that these cells subsets was perturbed on cytospin preparations of TECs, or that are exhausted or are end-stage cells that nevertheless may have a the antiserum/assay was less sensitive. The frequencies we report role as scaffold to support the thymus structure. It is plausible that in the present study were comparable in independent experiments the detrimental effects of either ablation of Foxn1 expression (by using our anti-HA or anti-Foxn1 mAb. conditional deletion of Foxn1-floxed alleles) or Foxn1-expressing The early “ubiquitous” expression of Foxn1 in the developing cells (by progressive intoxication of Foxn1-expressing cells) acted thymus is in line with the reported fate-mapping data (37, 38). We on the thymus only via the Foxn1+ TEC population. These data The Journal of Immunology 5685 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. rhFGF7 treatment generates CD802Ly51+Foxn1hi TECs. Six-day-old C57BL/6J mice were injected on three consecutive days with either PBS or 5 mg/kg rhFGF7. Thymic cellularity (A), TEC numbers (B), TEC proliferation (C), and Foxn1 expression by TECs (D) were analyzed 3 d (s, three independent experiments with three mice per treatment) or 6 d (n, three independent experiments with three mice per treatment) after the first injection (mean 6 SD). (E) Flow cytometric analysis of Foxn1 expression by thymic stroma (live, CD452; left) and the phenotype of Foxn12 (middle) and Foxn1+ (right) TECs (live, CD452EpCAM+) 6 d after the first injection. The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. One representative out of three experiments is shown. imply that Foxn12 TECs, despite their abundance in the adult castration (23) are well in line with previous publications. Re- thymus, are unable to sustain a functional thymus (24, 38, 41–43). generation after DEX treatment reestablished the frequency of Reduced Foxn1 expression has been linked to thymus aging Foxn1-expressing cells without changes in Foxn1 levels, and the and involution. Our detailed kinetics of frequencies and levels of regrowth of the thymus in response to DEX was characterized by Foxn1 expression reveal a surprisingly early decline in Foxn1 the appearance of a CD802Ly51+Foxn1+ population. expression at a juvenile age. The ubiquitous requirement for Foxn1 The long-lasting rhFGF7-induced regeneration (34) relied on the expression in all TECs appears to end at around 2 wk of age, and expansion of a different TEC subset than that involved in the hence in a phase when the thymus is about to enter steady-state transient regeneration observed after castration (32, 44), although function. In this scenario, lower levels of Foxn1 may set the each response was accompanied by an upregulation of Foxn1 stage for the later occurring involution; however, this famous process protein levels. A predominant cTEC-like Ly51+Foxn1+ population is most likely multifactorial. could be seen in response to DEX administration and rhFGF7 The thymus is known for its regenerative capacity in response to treatment, as well as in Rag2-deficient mice lacking mature thy- thymic insults and thymic stimulation. The observed disappear- mocytes. That the transient castration response involved a CD80+ ance of the CD80+Ly512 mTECs in response to DEX (28) and the Ly512Foxn1+ mTEC-like population clearly pointed toward two emergence of a prominent CD80+Ly512 mTEC population after different regenerative mechanisms. Concomitantly, Fgf7 deficiency 5686 Foxn1 PROTEIN IN SPACE AND TIME Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Castration generates CD80+Ly512Foxn1hi TECs. Nine- to eleven-week-old C57BL/6J untreated males (d), sham-castrated controls (N), and castrated males (▴) were analyzed 3, 8, and 14 d after surgery (n = 7–9 per treatment and time point). Thymocyte numbers are shown (A), as are the frequency (B) and levels of Foxn1 expression (C) by day 14 after surgery TECs (live, CD452EpCAM+; mean 6 SD). (D) Foxn1 levels expressed by thymic stroma (live, CD452; left), the expression of CD80 versus Ly51 on Foxn12 (middle), and Foxn1+ (right) TECs of sham-castrated (top panels) and castrated males (bottom panels) at 14 d after surgery. The indicated frequencies refer to the distribution of Foxn1+ and Foxn12 fractions among EpCAM+ TECs. One representative out of three experiments is shown. does not result in an inability to respond to castration (45). It seems Acknowledgments that each thymic injury, and thereby the regenerative outcome, has We thank Carmen Blum and Susan Schlenner for help with the generation its own characteristic responding TEC population. of mouse mutants; the Imaging Core Facility for help with immunofluores- Whether the predominant Ly51+Foxn1+ cTEC-like cells of cence; the German Cancer Research Center animal facility for expert mouse Rag2-deficient mice, as well as the phenotypically identical pop- husbandry; and Andrea Weber, Tabea Arnsperger, and Sven Scha¨fer for ulations expanding in response to DEX and rhFGF7, represent technical assistance. bona fide cTECs remains to be shown. It seems more likely that some TEC progenitor population may also display the Ly51+ Disclosures + Foxn1 phenotype, as does the vast majority of TECs in unborn The authors have no financial conflicts of interest. mice, and at least some of these cells have been demonstrated to exhibit progenitor function (46). Moreover, cTEC-specific b5t promoter–driven Cre labels mTECs, once more suggesting an References overlap between the phenotypes of TEC progenitors and cTECs 1. Nehls, M., D. Pfeifer, M. Schorpp, H. Hedrich, and T. Boehm. 1994. New (47). The appearance of cTEC markers on mTECs might represent member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature 372: 103–107. a coincidental, transient upregulation of these markers during 2. Frank, J., C. Pignata, A. A. Panteleyev, D. M. Prowse, H. Baden, L. Weiner, mTEC development, or it requires a revised order of TEC matu- L. Gaetaniello, W. Ahmad, N. Pozzi, P. B. Cserhalmi-Friedman, et al. 1999. ration, as suggested (48). Exposing the human nude phenotype. Nature 398: 473–474. 3. Schorpp, M., M. Hofmann, T. N. Dear, and T. Boehm. 1997. Characterization of In summary, our in-depth analysis of Foxn1 expression in TEC mouse and human nude . Immunogenetics 46: 509–515. subsets in the developing, aging, and regenerating thymus may 4. Schlake, T., M. Schorpp, M. Nehls, and T. Boehm. 1997. The nude gene en- codes a sequence-specific DNA binding protein with homologs in organisms provide a useful basis to address molecular functions of this that lack an anticipatory immune system. Proc. Natl. Acad. Sci. USA 94: 3842– transcription factor in the near future. 3847. The Journal of Immunology 5687

5. Schuddekopf,€ K., M. Schorpp, and T. Boehm. 1996. The whn transcription factor tolerance-inducing medullary thymic epithelial cells after cyclosporine, cyclo- encoded by the nude locus contains an evolutionarily conserved and functionally phosphamide, and dexamethasone treatment. J. Immunol. 183: 823–831. indispensable activation domain. Proc. Natl. Acad. Sci. USA 93: 9661–9664. 29. Sutherland, J. S., G. L. Goldberg, M. V. Hammett, A. P. Uldrich, S. P. Berzins, 6. Condie, N. R. M. B. G., and B. G. Condie. 2010. Transcriptional regulation of T. S. Heng, B. R. Blazar, J. L. Millar, M. A. Malin, A. P. Chidgey, and thymus organogenesis and thymic epithelial cell differentiation. Prog. Mol. Biol. R. L. Boyd. 2005. Activation of thymic regeneration in mice and humans fol- Transl. Sci. 92: 103–120. lowing androgen blockade. J. Immunol. 175: 2741–2753. 7. Itoi, M., H. Kawamoto, Y. Katsura, and T. Amagai. 2001. Two distinct steps of 30. Roden, A. C., M. T. Moser, S. D. Tri, M. Mercader, S. M. Kuntz, H. Dong, immigration of hematopoietic progenitors into the early thymus anlage. Int. A. A. Hurwitz, D. J. McKean, E. Celis, B. C. Leibovich, et al. 2004. Aug- Immunol. 13: 1203–1211. mentation of T cell levels and responses induced by androgen deprivation. J. 8. Nehls, M., B. Kyewski, M. Messerle, R. Waldschutz,€ K. Schuddekopf,€ Immunol. 173: 6098–6108. A. J. Smith, and T. Boehm. 1996. Two genetically separable steps in the dif- 31. Olsen, N. J., S. M. Viselli, K. Shults, G. Stelzer, and W. J. Kovacs. 1994. In- ferentiation of thymic epithelium. Science 272: 886–889. duction of immature thymocyte proliferation after castration of normal male 9. Bleul, C. C., T. Corbeaux, A. Reuter, P. Fisch, J. S. Mo¨nting, and T. Boehm. mice. Endocrinology 134: 107–113. 2006. Formation of a functional thymus initiated by a postnatal epithelial pro- 32. Goldberg, G. L., J. A. Dudakov, J. J. Reiseger, N. Seach, T. Ueno, K. Vlahos, genitor cell. Nature 441: 992–996. M. V. Hammett, L. F. Young, T. S. P. Heng, R. L. Boyd, and A. P. Chidgey. 2010. 10. Bleul, C. C., and T. Boehm. 2005. BMP signaling is required for normal thymus Sex steroid ablation enhances immune reconstitution following cytotoxic anti- development. J. Immunol. 175: 5213–5221. neoplastic therapy in young mice. J. Immunol. 184: 6014–6024. 11. Blackburn, C. C., C. L. Augustine, R. Li, R. P. Harvey, M. A. Malin, R. L. Boyd, 33. Kelly, R. M., S. L. Highfill, A. Panoskaltsis-Mortari, P. A. Taylor, R. L. Boyd, J. F. Miller, and G. Morahan. 1996. The nu gene acts cell-autonomously and is G. A. Holla¨nder, and B. R. Blazar. 2008. Keratinocyte growth factor and an- required for differentiation of thymic epithelial progenitors. Proc. Natl. Acad. drogen blockade work in concert to protect against conditioning regimen- Sci. USA 93: 5742–5746. induced thymic epithelial damage and enhance T-cell reconstitution after murine 12. Muller,€ S. M., G. Terszowski, C. Blum, C. Haller, V. Anquez, S. Kuschert, bone marrow transplantation. Blood 111: 5734–5744. P. Carmeliet, H. G. Augustin, and H.-R. Rodewald. 2005. Gene targeting of 34. Rossi, S. W., L. T. Jeker, T. Ueno, S. Kuse, M. P. Keller, S. Zuklys, VEGF-A in thymus epithelium disrupts thymus blood vessel architecture. Proc. A. V. Gudkov, Y. Takahama, W. Krenger, B. R. Blazar, and G. A. Holla¨nder. Natl. Acad. Sci. USA 102: 10587–10592. 2007. Keratinocyte growth factor (KGF) enhances postnatal T-cell development 13. Jenkinson, W. E., A. Bacon, A. J. White, G. Anderson, and E. J. Jenkinson. 2008. via enhancements in proliferation and function of thymic epithelial cells. Blood Downloaded from An epithelial progenitor pool regulates thymus growth. J. Immunol. 181: 6101– 109: 3803–3811. 6108. 35. Erickson, M., S. Morkowski, S. Lehar, G. Gillard, C. Beers, J. Dooley, 14. Ortman, C. L., K. A. Dittmar, P. L. Witte, and P. T. Le. 2002. Molecular char- J. S. Rubin, A. Rudensky, and A. G. Farr. 2002. Regulation of thymic epithelium acterization of the mouse involuted thymus: aberrations in expression of tran- by keratinocyte growth factor. Blood 100: 3269–3278. scription regulators in thymocyte and epithelial compartments. Int. Immunol. 14: 36. Itoi, M., N. Tsukamoto, and T. Amagai. 2007. Expression of Dll4 and CCL25 in 813–822. Foxn1-negative epithelial cells in the post-natal thymus. Int. Immunol. 19: 127–132. 15. Bredenkamp, N., S. Ulyanchenko, K. E. O’Neill, N. R. Manley, H. J. Vaidya, and 37. Liston, A., A. G. Farr, Z. Chen, C. Benoist, D. Mathis, N. R. Manley, and

C. C. Blackburn. 2014. An organized and functional thymus generated from A. Y. Rudensky. 2007. Lack of Foxp3 function and expression in the thymic http://www.jimmunol.org/ FOXN1-reprogrammed fibroblasts. Nat. Cell Biol. 16: 902–908. epithelium. J. Exp. Med. 204: 475–480. 16. Soza-Ried, C., C. C. Bleul, M. Schorpp, and T. Boehm. 2008. Maintenance of 38. Corbeaux, T., I. Hess, J. B. Swann, B. Kanzler, A. Haas-Assenbaum, and thymic epithelial phenotype requires extrinsic signals in mouse and zebrafish. J. T. Boehm. 2010. Thymopoiesis in mice depends on a Foxn1-positive thymic Immunol. 181: 5272–5277. epithelial cell lineage. Proc. Natl. Acad. Sci. USA 107: 16613–16618. 17. Srinivas, S., T. Watanabe, C. S. Lin, C. M. William, Y. Tanabe, T. M. Jessell, and 39. Ucar, A., O. Ucar, P. Klug, S. Matt, F. Brunk, T. G. Hofmann, and B. Kyewski. F. Costantini. 2001. Cre reporter strains produced by targeted insertion of EYFP 2014. Adult thymus contains FoxN12 epithelial stem cells that are bipotent for and ECFP into the ROSA26 locus. BMC Dev. Biol. 1: 4. medullary and cortical thymic epithelial lineages. Immunity 41: 257–269. 18. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, 40. Dooley, J., M. Erickson, and A. G. Farr. 2009. Lessons from thymic epithelial J. Charron, M. Datta, F. Young, A. M. Stall, et al. 1992. RAG-2-deficient mice heterogeneity: FoxN1 and tissue-restricted by extrathymic, lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell endodermally derived epithelium. J. Immunol. 183: 5042–5049. 68: 855–867. 41. Guo, J., Y. Feng, P. Barnes, F.-F. Huang, S. Idell, D.-M. Su, and H. Shams. 2012.

19. Rode, I., and T. Boehm. 2012. Regenerative capacity of adult cortical thymic Deletion of FoxN1 in the thymic medullary epithelium reduces peripheral T cell by guest on September 25, 2021 epithelial cells. Proc. Natl. Acad. Sci. USA 109: 3463–3468. responses to infection and mimics changes of aging. PLoS One 7: e34681. 20. Gereke, M., A. Autengruber, L. Gro¨be, A. Jeron, D. Bruder, and S. Stegemann- 42. Cheng, L., J. Guo, L. Sun, J. Fu, P. F. Barnes, D. Metzger, P. Chambon, Koniszewski. 2012. Flow cytometric isolation of primary murine type II alveolar R. G. Oshima, T. Amagai, and D. M. Su. 2010. Postnatal tissue-specific dis- epithelial cells for functional and molecular studies. J. Vis. Exp. 2012: e4322. ruption of transcription factor FoxN1 triggers acute thymic atrophy. J. Biol. 21. Terszowski, G., S. M. Muller,€ C. C. Bleul, C. Blum, R. Schirmbeck, J. Reimann, Chem. 285: 5836–5847. L. D. Pasquier, T. Amagai, T. Boehm, and H. R. Rodewald. 2006. Evidence for a 43. Guo, J., M. Rahman, L. Cheng, S. Zhang, A. Tvinnereim, and D.-M. Su. 2011. functional second thymus in mice. Science 312: 284–287. Morphogenesis and maintenance of the 3D thymic medulla and prevention of 22. Chen, L., S. Xiao, and N. R. Manley. 2009. Foxn1 is required to maintain the nude skin phenotype require FoxN1 in pre- and post-natal K14 epithelium. J. postnatal thymic microenvironment in a dosage-sensitive manner. Blood 113: Mol. Med. 89: 263–277. 567–574. 44. Griffith, A. V., M. Fallahi, T. Venables, and H. T. Petrie. 2012. Persistent de- 23. Gray, D. H. D., N. Seach, T. Ueno, M. K. Milton, A. Liston, A. M. Lew, generative changes in thymic organ function revealed by an inducible model of C. C. Goodnow, and R. L. Boyd. 2006. Developmental kinetics, turnover, and organ regrowth. Aging Cell 11: 169–177. stimulatory capacity of thymic epithelial cells. Blood 108: 3777–3785. 45. Goldberg, G. L., O. Alpdogan, S. J. Muriglan, M. V. Hammett, M. K. Milton, 24. Sun, L., J. Guo, R. Brown, T. Amagai, Y. Zhao, and D.-M. Su. 2010. Declining J. M. Eng, V. M. Hubbard, A. Kochman, L. M. Willis, A. S. Greenberg, et al. expression of a single epithelial cell-autonomous gene accelerates age-related 2007. Enhanced immune reconstitution by sex steroid ablation following allo- thymic involution. Aging Cell 9: 347–357. geneic hemopoietic stem cell transplantation. J. Immunol. 178: 7473–7484. 25. Zook, E. C., P. A. Krishack, S. Zhang, N. J. Zeleznik-Le, A. B. Firulli, 46. Baik, S., E. J. Jenkinson, P. J. L. Lane, G. Anderson, and W. E. Jenkinson. 2013. P. L. Witte, and P. T. Le. 2011. Overexpression of Foxn1 attenuates age- Generation of both cortical and Aire+ medullary thymic epithelial compartments associated thymic involution and prevents the expansion of peripheral CD4 from CD205+ progenitors. Eur. J. Immunol. 43: 589–594. memory T cells. Blood 118: 5723–5731. 47. Ohigashi, I., S. Zuklys, M. Sakata, C. E. Mayer, S. Zhanybekova, S. Murata, 26. Bredenkamp, N., C. S. Nowell, and C. C. Blackburn. 2014. Regeneration of the K. Tanaka, G. A. Holla¨nder, and Y. Takahama. 2013. Aire-expressing thymic aged thymus by a single transcription factor. Development 141: 1627–1637. medullary epithelial cells originate from b5t-expressing progenitor cells. Proc. 27. Kong, F.-K., C.-L. H. Chen, and M. D. Cooper. 2002. Reversible disruption of Natl. Acad. Sci. USA 110: 9885–9890. thymic function by steroid treatment. J. Immunol. 168: 6500–6505. 48. Alves, N. L., Y. Takahama, I. Ohigashi, A. R. Ribeiro, S. Baik, G. Anderson, and 28. Fletcher, A. L., T. E. Lowen, S. Sakkal, J. J. Reiseger, M. V. Hammett, N. Seach, W. E. Jenkinson. 2014. Serial progression of cortical and medullary thymic H. S. Scott, R. L. Boyd, and A. P. Chidgey. 2009. Ablation and regeneration of epithelial microenvironments. Eur. J. Immunol. 44: 16–22.