Thymic Epithelial Cells Ontogeny and Regulation of IL-7-Expressing

Ontogeny and Regulation of IL-7-Expressing Thymic Epithelial Cells Monica Zamisch, Billie Moore-Scott, Dong-ming Su, Philip J. Lucas, Nancy Manley and Ellen R. Richie This information is current as of September 30, 2021. J Immunol 2005; 174:60-67; ; doi: 10.4049/jimmunol.174.1.60 http://www.jimmunol.org/content/174/1/60 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ontogeny and Regulation of IL-7-Expressing Thymic Epithelial Cells1

Monica Zamisch,* Billie Moore-Scott,† Dong-ming Su,† Philip J. Lucas,‡ Nancy Manley,† and Ellen R. Richie2*

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identiﬁed IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7- Downloaded from expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1؊/؊ mice are developmentally and functionally distinct. The Journal of Immunology, 2005, 174: 60–67. http://www.jimmunol.org/

␣Ϫ/Ϫ ␥ Ϫ/Ϫ nterleukin-7 is a pleiotrophic cytokine that is produced by effector Bax rescues T cell development in IL-7R and c thymic and bone marrow stromal cells and is essential for T mice (8–10). IL-7 signaling also promotes the survival of DN thy- and B cell lymphopoiesis (reviewed in Refs. 1 and 2). The mocytes undergoing transition through the ␤-selection checkpoint I ϩ ϩ IL-7R consists of an ␣-chain that is also a component of the thymic to the CD4 CD8 double-positive (DP) stage (11) and prolifera- stromal lymphopoietin receptor, and the common cytokine recep- tion of positively selected CD4ϩCD8Ϫ and CD4ϪCD8ϩ single- ␥ ␥ 3 tor -chain ( c) that is present in IL-2, -4, -9, -15, and -21 recep- positive (SP) thymocytes (12). In addition to enhancing the sur- tors (3). Signaling through the IL-7R initiates multiple signaling vival and proliferation of ␣␤-lineage thymocytes, IL-7 regulates cascades, including activation of protein tyrosine kinases Jak1 and TCR␥ gene rearrangement by controlling locus accessibility, and by guest on September 30, 2021 ␣ Jak3 that associate with the intracellular domains of the IL-7R is, therefore, essential for the development of ␥␦ T cells (13–15). and ␥ chains, respectively. Activated Jak1 and Jak3 phosphorylate c Although recent studies suggest a less stringent requirement for STAT1 and STAT5, resulting in altered gene expression patterns IL-7 in promoting survival of fetal compared with adult thymo- (4). IL-7-mediated signals modulate the expression of genes that cytes (13, 16), IL-7 expression has been reported in the early fetal affect thymocyte survival, proliferation, and differentiation. Dis- Ϫ/Ϫ ␣Ϫ/Ϫ ␥ Ϫ/Ϫ thymus (17, 18). Intrathymic production of IL-7 is primarily a ruption of IL-7 signaling in IL-7 , IL-7R , and c mice severely reduces thymic cellularity and impairs thymocyte devel- function of thymic epithelial cells (TECs) (19, 20). The TEC com- opment (5–7). This phenotype is due, in part, to reduced survival partment is heterogeneous, consisting of subcapsular, cortical, and of CD4ϪCD8Ϫ double-negative (DN) thymocyte progenitors be- medullary subsets defined by morphological properties, antigenic cause introduction of a bcl-2 transgene or deletion of the death profiles, and keratin expression patterns (reviewed in Refs. 21 and 22). However, because prior studies analyzed IL-7 expression by RT-PCR, the relative localization of TEC subsets that express IL-7 *Science Park-Research Division, University of Texas M.D. Anderson Cancer Cen- in the fetal and adult thymic microenvironment was not deter- ter, Smithville, TX 78957, and University of Texas Graduate School of Biomedical mined. Furthermore, the earliest developmental stage at which Sciences, Houston, TX 77225; †Department of Genetics, University of Georgia, Ath- ens, GA 30602; and ‡Experimental Immunology Branch, National Cancer Institute, IL-7 is expressed in the thymic primordium has not been estab- Bethesda, MD 20892 lished. Given the functional significance of IL-7 signaling and the Received for publication March 9, 2004. Accepted for publication October 18, 2004. compartmentalization of thymocyte and epithelial subsets, we per- The costs of publication of this article were defrayed in part by the payment of page formed in situ hybridization (ISH) analyses to explore the emer- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. gence, distribution, and regulation of IL-7-expressing TECs during ontogeny and in postnatal mice. The data show striking differences 1 These studies were generously supported by a grant from The Fant Foundation. This work was also supported by National Institutes of Health Grant AI041543, National in the localization and frequency of IL-7 expression in fetal and Institute on Environmental Health Sciences Center Grant ES07784 (to E.R.), and adult TECs. In addition, examination of IL-7 expression in the National Institutes of Health Grant AI055001 (to N.M.). third pharyngeal pouch endoderm of wild-type and nude mice re- 2 Address correspondence and reprint requests to Dr. Ellen Richie, P.O. Box 389, Science Park-Research Division, Smithville, TX 78957. E-mail address: veals IL-7 to be an early marker of TEC fate. Finally, differences [email protected] in the thymocyte subsets that regulate IL-7 expression patterns 3 ␥ ␥ Ϫ/Ϫ Abbreviations used in this paper: c, common -chain; CLP, common lymphoid demonstrate that cortical TECs in RAG-1 mice are develop- progenitor; CMJ, corticomedullary junction; DN, double negative; DP, double positive; E, embryonic day; ECM, extracellular matrix; ISH, in situ hybridization; K5, mentally and functionally immature compared with the wild-type keratin 5; SP, single positive; TEC, thymic epithelial cell. cortical TEC compartment.

Materials and Methods scopic analysis was performed with an Olympus ProVis AX70 microscope Mice and tissue preparation (Olympus). C57BL/6J, RAG-1Ϫ/Ϫ, and ␯/J mice were purchased from The Jackson Thymic suspensions and FACS sorting Laboratory. RAG-2Ϫ/Ϫ/␥ Ϫ/Ϫ mice were purchased from Taconic Farms. c Thymocytes and TECs were released from E12.5 thymi by trypsin diges- Ikaros-null mice were the generous gift of K. Georgopoulos (Harvard Med- tion, as previously described (28). Single cell suspensions were stained ical School, Charlestown, MA) (23). Embryos were obtained by setting up with allophycocyanin-conjugated anti-CD45 Ab (clone 30-F11) (BD Bio- timed matings for 16 h and considering the morning of finding the vaginal sciences). Hemopoietic and stromal cells were isolated by sorting the Ab- plug as embryonic day 0.5 (E0.5). Embryos were fixed in Bouin’s solution ϩ Ϫ stained cells for CD45 (Ͼ98% purity) and CD45 (Ͼ94% purity) sub- for 3–5 h; washed overnight in 70% ethanol; dehydrated in series of 30, 50, sets, respectively, using a Corixa Elite flow cytometer. 70, 90, and 100% ethanol washes; and paraffin embedded. Thymi from postnatal mice were obtained by dissection and fixed as above, except that the dehydration protocol used 70, 90, and 100% ethanol. For whole mount Results in situ hybridization, embryos were fixed in 4% paraformaldehyde/PBST IL-7 expression is restricted to the thymus domain of the early overnight, washed twice in PBST, and dehydrated in series of 30, 50, 70, primordium 90, and 100% methanol. IL-7 is a secreted cytokine that binds to MHC class II-positive In situ hybridization TECs and fibroblasts in a heparin sulfate-dependent manner (29). Paraffin section ISH was performed, as previously described (24), with the To avoid detecting cells that bind, but do not synthesize IL-7, we exception that the proteinase K digestion was omitted. The IL-7 riboprobe evaluated IL-7 mRNA expression by ISH analysis. Although IL-7 was obtained by RT-PCR on RNA from the cell line mouse plasmacytoma expression was not detected in the pharyngeal region at E10.5 line J558 obtained from American Type Culture Collection (Manassas, (data not shown), IL-7 message is expressed in the third pharyn- Downloaded from VA). The PCR used the following oligos: 5Ј oligo, 5Ј-GCGACTGGATC CGACTACACCCACCTCCCGCAGACC-3Ј;3Ј oligo, 5Ј-CGATCCGG geal pouch endoderm by E11.5. Fig. 1 shows that IL-7 expression ATCCAAGATTCTTGGAGGTTGTTACTAC-3Ј. is restricted to the epithelium of the third pouch and is not detected The 5Ј oligo ends right before the ATG site, and the 3Ј oligo starts 30 in other pharyngeal compartments. At E11.5, the parathyroid- and bp downstream from the stop codon. After adding BamHI sites to each end, ϩ thymus-specific domains of the third pouch are contiguous within the 546-bp fragment was cloned into the blunted EcoRI site of BSII SK a common primordium that has not yet separated from the pharynx (Strategene). The 534-bp BamHI fragment was sequenced using T3 and T7 primers of BS. (30). A higher magnification view (Fig. 1B) shows that IL-7 ex- http://www.jimmunol.org/ The Gcm2 probe was generated by PCR amplification, as described (25). pression is restricted to the ventral aspect of the third pouch, the The Foxn1 probe was PCR amplified from E11.5 mouse cDNA using prim- domain that is fated to develop into thymus as opposed to para- Ј ers encompassing the eighth and ninth exons and 3 UTR (26). The IL-7, thyroid (31). Although IL-7 plays an essential role in thymocyte Foxn1, and Gcm2 riboprobes (sense and antisense) were labeled with digoxigenin during in vitro transcription. BM purple (Roche) was used as development, the early appearance of IL-7-expressing TECs dur- a chromagen for detection of the hybridized probe, and the sections were ing ontogeny suggested that it may function to stimulate TEC pro- counterstained with nuclear fast red. Whole mount ISH was performed, as genitors via an autocrine pathway. To explore this possibility, we described (27). assessed IL-7R␣ expression on stromal as well as hemopoietic cells obtained from fetal thymi. Single cell suspensions of E12

Semiquantitative RT-PCR by guest on September 30, 2021 thymi were stained with anti-CD45 and sorted to isolate CD45Ϫ Wild-type and nude E12 fetal thymic lobes were separately collected in stromal cells and CD45ϩ hemopoietic cells. Consistent with earlier TRIzol (Invitrogen Life Technologies) and homogenized using Micro Pel- reports showing that IL-R␣ is expressed on fetal thymocyte pro- let Pestles (Nalge Nunc International). RNA was isolated, the genomic ϩ DNA was depleted with DNase I (Invitrogen Life Technologies), and. re- genitors (32–34), RT-PCR analysis of CD45 cells revealed a verse transcription of total RNA to cDNA was performed using Super- Script II (Invitrogen Life Technologies), following manufacturer’s protocol. Equal amounts of cDNA from each strain were added to a final 20 ␮l PCR mix using the Qiagen TaqPCR kit. PCR conditions for IL-7 and Foxn1 were: initial denaturation of 94°C 3 min; 25–35 cycles of 94°C 0.5–1 min, 55–70°C 0.5–1 min, and 72°C 1 min; and final extension of 72°C 10 min. PCR conditions for IL-7R␣ were initial denaturation of 95°C 1 min; 40 cycles of 95°C 30 min, 55°C 40 min, 72°C 45 min; and final extension of 72°C 10 min. PCR conditions for actin were: initial denaturation of 95°C 5 min; 35 cycles of 95°C 1 min, 56°C 1 min, 72°C 3 min; and final extension of 72°C 10 min. PCR products were visualized by 5% acrylamide gel electrophoresis and ethidium bromide staining. Band den- sities were measured and analyzed using the Molecular Analyst software (Bio-Rad, version 1.4.1). Primer sequences were: IL-7 (5Ј), 5Ј-ACT ACA CCC ACC TCC CGC A-3Ј; IL-7 (3Ј), 5Ј-TCT CAG TAG TCT CTT TAG G-3Ј; GADPH (5Ј), 5Ј-GTC TAC ATG TTC CAG TAT GAC TCC ACT CAC-3Ј; GADPH (3Ј), 5Ј-CAA TCT TGA GTG AGT TGT CAT ATT TCT CGT-3Ј; IL-7R␣ (5Ј), 5Ј-GAC ATC AGA ATT CTT ACT GAT TGG-3Ј; IL-7R␣ (3Ј), 5Ј-GGC GAG CTC GCC TTC GGG AAT GAA ACT CAC AT-3Ј; actin (5Ј), 5Ј-GTT TGA GAC CTT CAA CAC C-3Ј; actin (3Ј), 5Ј-GTG GCC ATC CCT GCT CGA AGT C-3Ј. Immunohistology FIGURE 1. IL-7 expression in the third pharyngeal pouch of E11.5 C57BL/6J embryo. In situ hybridization analysis of IL-7 expression in a Paraffin-embedded tissue sections were deparaffinized and rehydrated in sagittal section of an E11.5 embryo. A, IL-7-expressing cells are localized 100–75% ethanol. OCT-embedded frozen tissue sections were air dried 30 in the third pharyngeal pouch. Numbers designate each pharyngeal pouch. min before acetone fixation. Sections were incubated overnight at 4°C with Dorsal is left; anterior is up. B, A high magnification image of the third optimal dilutions of polyclonal anti-mouse keratin 5 (K5; Covance Re- search) and/or monoclonal anti-mouse c-kit (BD Pharmingen). Immunore- pouch shows that IL-7-expressing cells are present in the ventral aspect of activity to c-kit was enhanced by tyramide amplification (PerkinElmer Life the third pouch in the common thymus/parathyroid primordium. C, RT- Sciences). Controls included slides incubated with nonimmune rabbit- PCR analysis demonstrates that IL-7R␣ expression is detected in FACS- ϩ Ϫ matched Ig or isotype-matched mouse Ig. For costaining, sections were sorted CD45 hemopoietic cells, but not in CD45 stromal cells from E12 incubated simultaneously with primary Abs from different species. Micro- fetal thymi. Two dilutions of each cDNA were used for PCR. 62 IL-7-EXPRESSING TECs strong IL-7R␣ signal (Fig. 1C). In contrast, IL-7R␣ expression was not detected in the CD45Ϫ compartment. Similar results were obtained with CD45ϩ and CD45Ϫ cells from E14.5 embryos (data not shown). These data indicate that IL-7 expression in the early thymic primordium promotes development of thymocyte precursors, but does not function as an autocrine factor for TEC progenitors. The IL-7 expression pattern at E11.5 was similar to that previously described for Foxn1 (31). Foxn1 is a forkhead class transcription factor that is expressed in the thymic primordium by E11.25 and is required for TEC development. Nude mice are ho- mozygous for a null mutation in Foxn1, resulting in defective hair and thymus development due to impaired epithelial differentiation in skin and thymus (31, 35, 36). TECs in the nude thymus are arrested at a stage corresponding to an early progenitor cell type, suggesting that TECs are specified to a thymus fate, but fail to FIGURE 3. IL-7 is expressed in the third pouch endoderm of nude mice. A, ISH analysis of IL-7 expression in a transverse section of an E12 differentiate (35, 37, 38). Comparative whole mount ISH analysis nude embryo shows that IL-7 expression is independent of Foxn1 expres- shows that Foxn1 and IL-7 are expressed in overlapping domains sion. B, A higher magnification image of IL-7-expressing cells in the third at E11.5 (Fig. 2, A and B). In this comparison, the IL-7 domain pharyngeal pouch. C, RT-PCR analysis confirms that IL-7 is expressed in Downloaded from appears slightly smaller than the Foxn1 domain, although devel- wild-type and nude thymi from E12 embryos. opment at this stage is rapid, and this could simply represent slight differences in the developmental stages of the embryos, or in the exact plane of the section. In contrast, Gcm2 is a transcription Diminished frequency and restricted localization of IL-7- factor that is expressed specifically in the parathyroid-specific do- expressing TECs during ontogeny main of the third pouch endoderm (31). Comparison of the IL-7 http://www.jimmunol.org/ and Gcm2 expression patterns at E11.5 confirmed that IL-7 ex- As shown in Fig. 4, IL-7 is abundantly expressed throughout the pression is restricted to the ventral and distal thymus-specific do- E12.5 and E13.5 wild-type fetal thymus. By E15.5, there is a no- main of the developing primordium (Fig. 2, B and C). table increase in thymic size and cellularity due to thymocyte and TEC proliferation. Thymic expansion is accompanied by a de- creased frequency of IL-7-producing TECs particularly in the cen- Foxn1 does not regulate IL-7 expression tral region of the thymus. As development proceeds, the distribu- Because Foxn1 is required for TEC differentiation and is expressed tion of IL-7-expressing TECs becomes increasingly restricted such within a similar developmental time frame as IL-7, we considered that by E16.5, IL-7-expressing TECs are found primarily toward that IL-7 might be a downstream target of Foxn1-mediated tran- the subcapsular region. In the neonatal thymus, IL-7 expression by guest on September 30, 2021 scriptional regulation. To explore this possibility, we examined remains prominent in the subcapsular zone and is apparent at the IL-7 expression in the rudimentary thymic remnant of early em- junction between the cortex and the developing medullary regions bryos from nude mice. Fig. 3, A and B, shows that IL-7 expression (Fig. 5). The relatively low frequency of IL-7-expressing cells in is readily detected by ISH analysis in the thymic primordium of the cortex could reflect down-regulation of IL-7 expression in cor- E12 nude mice. Semiquantitative RT-PCR analysis confirmed the tical TECs or relocalization of IL-7-expressing TECs along with presence of IL-7 message in the E12 nude thymus anlage, although expansion of an IL-7-negative subset. The paucity of IL-7-express- a stronger signal was observed in wild-type compared with nude ing cells in the cortex is even more striking in 1-wk postnatal and fetal thymi (Fig. 3C). These results demonstrate that induction of adult thymi in which IL-7-expressing cells are concentrated in the IL-7 expression is independent of Foxn1-mediated regulation dur- corticomedullary junction (CMJ) and medulla. Expression of IL-7 ing thymic organogenesis. Furthermore, the presence of IL-7 mes- in the medullary region is consistent with earlier reports showing sage in the absence of functional Foxn1 establishes IL-7 as an that IL-7 promotes the proliferation and differentiation of SP thy- early developmental marker of endodermal commitment to a thymocytes (12, 39). IL-7-expressing cells in the postnatal thymus are mic epithelial cell fate. also associated with large blood vessels that are characteristically

FIGURE 2. IL-7 expression is restricted to the thymus-specific portion of the shared parathyroid-thymus primordium. Images shown are the inside FIGURE 4. Changes in the frequency and localization of IL-7-express- surface of E11.5 embryos hemisected parasagittally and stained in whole ing cells during ontogeny. Top row, Shows low magnification images, and mount by in situ hybridization. The developing third pouch primordium is bottom row shows high magnification images of ISH analyses of IL-7- outlined in each panel. Dorsal is left; anterior is up. A, Foxn1 is expressed expressing cells in transverse sections of thymi from embryos at the indi- in the ventral primordium. B, IL-7 expression is similar to Foxn1. C, Gcm2 cated ages. There is a notable decrease in the frequency of IL-7-expressing expression marks the dorsal, parathyroid-specific domain. cells that are localized near the subcapsular region by E16.5. The Journal of Immunology 63

FIGURE 5. The majority of IL-7-expressing cells are localized at the CMJ and in the medulla of postnatal thymi. A, ISH analysis of IL-7 expression in the newborn thymus shows positive cells in the subcapsular region and outlining the medullary regions. B, A consecutive H&E-stained section of the same newborn thymus delineates cortical and medullary regions. C, By 1 wk of age, IL-7-expressing cells are found throughout the medullary region and surround large vessels at the CMJ. D, A higher magnification image of the boxed area in C shows IL-7-expressing cells lining FIGURE 6. Thymocyte-derived signals are not necessary for induction Downloaded from large vessels. E, The majority of IL-7-expressing cells are found in the of IL-7 expression in the early fetal thymus. A, Cryostat sections of E13.5 ␥ medulla and corticomedullary junction of a 4-wk-old thymus hybridized wild-type C57BL6/J, RAG2/ c-deficient, and Ikaros null embryos were with an antisense IL-7 probe. F, No positive cells are present in medulla or stained with anti-K5 and anti-c-kit. Reactivity with anti-K5 was detected cortex of a consecutive section hybridized with a sense IL-7 probe. with Texas Red-conjugated anti-Ig, and a tyramide amplification system was used to detect c-kit FITC staining. B, The top row shows low magnification images, and the bottom row shows high magnification images of

IL-7-expressing cells in transverse sections of thymi from E13.5 wild-type, http://www.jimmunol.org/ ␥ Ϫ/Ϫ found in the perimedullary region at the CMJ (Fig. 5D). Hemo- RAG-2/ c , and Ikaros null embryos. poietic progenitors gain entry from the bloodstream into the postnatal thymus by extravasation at these sites (40, 41). The perivascular localization of IL-7-expressing cells suggests that as are essential for proper organization and development of cortical thymocyte progenitors enter the postnatal thymus, they encounter and medullary TEC subsets during late fetal development and in high concentrations of IL-7. the adult (42, 44–46). To determine whether thymocytes are also required to regulate IL-7 expression in the postnatal thymic epi- Initiation of IL-7 expression in the fetal thymus is independent thelial compartment, we performed a comparative ISH analysis of of thymocyte-derived signals ␥ Ϫ/Ϫ by guest on September 30, 2021 IL-7 expression in adult thymi from wild-type, RAG-2/ c , and We previously reported that hemopoietic-derived inductive signals RAG-1Ϫ/Ϫ mice. In contrast to the developmental block at the ␥ Ϫ/Ϫ are not required to establish initial patterning of fetal TEC subsets DN2 stage in RAG-2/ c mice, thymocyte development is ar- as determined by expression of distinct keratin species and anti- rested at the DN3 stage in RAG-1Ϫ/Ϫ mice (43, 47). genic profiles (42). To determine whether hemopoietic-derived Fig. 7 shows that, as noted above, IL-7-expressing TECs are signals are required to initiate IL-7 expression in the thymic rudi- predominantly localized in the medullary region of the wild-type ment, we examined IL-7-expressing TECs in two experimental adult thymus, whereas the majority of cortical TECs do not express models that sustain early blocks in T cell development. The Ikaros detectable levels of IL-7 message. K5 is expressed by TECs in the transcription factor plays an essential role in commitment of he- medulla and at the corticomedullary junction, but not by cortical mopoietic stem cells to the lymphoid lineage. Targeted deletion of TECs (45). An analysis of K5 expression in a serial section con- the C-terminal region of the Ikaros gene results in a null phenotype firmed the overlap in localization of IL-7 and K5 expression in the characterized by failure of B and NK development throughout life and an absence of T cell precursors during the early fetal period (23). In contrast, thymocyte development is arrested at the c-kitϩCD25ϩ DN2 differentiation stage in mice that are deficient ␥ for both RAG-2 and the c receptor polypeptide chain (RAG2/ ␥ Ϫ/Ϫ c ) (43). As shown in Fig. 6A, fetal thymi from E13.5 RAG2/ ␥ Ϫ/Ϫ c mice contain few c-kit-positive thymocyte progenitors, and such cells are undetectable in E13.5 IkarosϪ/Ϫ thymi. Neverthe- less, Fig. 6B shows that IL-7 is abundantly expressed in E13.5 fetal ␥ Ϫ/Ϫ Ϫ/Ϫ thymi from both RAG2/ c and Ikaros mice. Furthermore, the localization pattern of IL-7-expressing TECs was comparable to that observed in wild-type controls. Therefore, the induction of IL-7 expression in fetal TECs occurs independently of thymocyte- derived signals.

Thymocyte-derived signals are required to regulate IL-7 FIGURE 7. IL-7 and K5 are differentially regulated in thymi of adult Ϫ/Ϫ ␥ Ϫ/Ϫ expression in the postnatal thymus wild-type, RAG-1 , and RAG-2/ c mice. Top row, Shows ISH analyses of IL-7-expressing cells. Bottom row, Consecutive sections are stained Although thymocyte/TEC interactions are not required to specify with anti-K5 and developed with Texas Red-conjugated anti-Ig. IL-7 and ␥ Ϫ/Ϫ phenotypic and functional characteristics of the thymic epithelial K5 expression are found throughout the RAG-2/ c thymus. K5 expres- compartment during early ontogeny, thymocyte-derived signals sion, but not IL-7 expression, is down-regulated in the RAG-1Ϫ/Ϫ thymus. 64 IL-7-EXPRESSING TECs wild-type thymus. In striking contrast, IL-7-expressing TECs are Ϫ/Ϫ ␥ Ϫ/Ϫ present throughout the RAG-1 and RAG-2/ c thymi. The ␥ Ϫ/Ϫ widespread IL-7 expression pattern in the adult RAG-2/ c thymus is consistent with the fact that the epithelial compartment in ␥ Ϫ/Ϫ the severely hypoplastic RAG-2/ c thymus has an abnormal two-dimensional organization and consists of immature TECs that coexpress K8 and K5 (42). K5 immunostaining of a serial section ␥ Ϫ/Ϫ veriﬁes that the majority of TECs in the RAG-2/ c thymus are K5 positive. In contrast, expression of IL-7 throughout the adult RAG-1Ϫ/Ϫ thymus was unexpected because we previously demonstrated that thymocytes blocked at the DN3 stage could signal cortical TEC progenitors to down-regulate K5 expression and or- ganize into a three-dimensional structure (45). These ﬁndings suggest that distinct thymocyte-derived signals control IL-7 expression and K5 down-regulation in the RAG-1Ϫ/Ϫ cortex and reveal that TECs in the cortex of RAG-1Ϫ/Ϫ mice are developmentally and functionally distinct from wild-type cortical TECs. Ϫ/Ϫ ␤

IL-7 expression pattern in RAG-2 TCR transgenic thymus Downloaded from To further examine the possibility that thymocyte-derived signals regulate the IL-7 expression pattern in the adult thymic cortex, we performed ISH analysis for IL-7 on thymi obtained from RAG- 2Ϫ/Ϫ mice that express a TCR␤ transgene. Introduction of a pro- ductively rearranged TCR␤ transgene into RAG-2-deficient mice restores the ability of DN3 thymocytes to undergo ␤-selection and http://www.jimmunol.org/ differentiate to the DP stage (48). Although RAG-2Ϫ/ϪTCR␤ϩ DN thymocytes traverse the ␤-selection checkpoint, the absence of TCR␣␤ on DP thymocytes precludes positive selection and maturation to the SP stage. As expected, an increase in thymic cellularity and the appearance of DP thymocytes were observed in RAG-2Ϫ/ϪTCR␤ϩ mice (Fig. 8). In contrast to the consistent pattern of IL-7 expression found in the RAG-1Ϫ/Ϫ thymus, IL-7- FIGURE 8. Expression of a TCR␤ transgene in RAG-2Ϫ/Ϫ thymocytes expressing TECs are primarily concentrated in the subcapsular re- results in thymic expansion, generation of DP thymocytes, and diminished gion of RAG-2Ϫ/ϪTCR␤ϩ mice. Furthermore, there is a notable frequency of IL-7-expressing TECs. A, ISH analysis of IL-7-expressing by guest on September 30, 2021 Ϫ/Ϫ Ϫ/Ϫ ␤ϩ decrease in the frequency of IL-7-expressing TECs in the ex- cells in RAG-2 and RAG-2 TCR adult thymi. B, Flow cytometric Ϫ/Ϫ panded cortex, a phenotype that is similar to the paucity of IL-7- analysis of CD4 and CD8 expression on thymocytes from RAG-2 and RAG-2Ϫ/ϪTCR␤ϩ adult thymi. expressing TECs observed in the wild-type cortex. Taken together, these data support the notion that progression through the ␤-selection checkpoint renders thymocytes competent to induce cortical expansion and alter the pattern of IL-7 expression in the cor- domain. This may reflect maturation-dependent constraints on the tical TEC compartment. level of IL-7 expression or the existence of distinct epithelial lineages. Further studies of precursor-progeny relationships and lin- Discussion eage analysis are necessary to distinguish these alternative This work presents four major findings concerning the appearance explanations. and regulation of IL-7-expressing TECs. First, IL-7 expression is Although there are notable similarities in the temporal and spa- an early marker of thymic epithelial fate. Second, IL-7-expressing tial expression of IL-7 and Foxn1 during fetal thymic develop- TECs are present throughout the early thymic rudiment, but are ment, the presence of IL-7 mRNA in the nude thymic rudiment organized into discrete zones in the adult thymus. Third, although demonstrates that Foxn1 transcriptional activity is not required for hemopoietic cells are not required to initiate IL-7 expression dur- IL-7 gene expression. The reduced IL-7 signal intensity in RT- ing thymic organogenesis, thymocyte-derived signals are neces- PCR analysis of nude compared with wild-type fetal thymi sug- sary to maintain a normal pattern of IL-7-expressing TECs in the gests that Foxn1 may modulate the level of IL-7 expression. How- adult thymus. Finally, the early arrest in thymocyte differentiation ever, this interpretation is subject to the caveat that wild-type and in RAG-1Ϫ/Ϫ mice results in a developmentally immature cortical nude thymi are not strictly comparable because thymic epithelial TEC compartment as reflected by the widespread distribution of differentiation is arrested in nude mice and, therefore, the lower IL-7-expressing TECs. signal may be an indirect consequence of impaired TEC differen- IL-7 expression is initiated between E10.5 and E11.5 in the tiation. Regardless, the Foxn1-independent expression of IL-7 in pharyngeal pouch endoderm of the common thymus/parathyroid thymic fated epithelial cells of the common primordium identifies primordium, specifically in the domain that is fated to develop into IL-7 as the earliest known marker of TEC fate in thymic ontogeny. the thymic epithelial compartment. Thus, IL-7 expression can be The early appearance of IL-7 message suggested that cross talk considered as a functional marker of early TECs, and is likely to between epithelial and hemopoietic progenitors might not be re- be expressed in the recently described K8ϩK5ϩMTS24ϩ progen- quired to initiate IL-7 expression in the fetal thymus. This premise itors that give rise to cortical and medullary epithelial compart- was supported by ISH data showing that high levels of IL-7 are ␥ Ϫ/Ϫ Ϫ/Ϫ ments (37, 38). Despite its early appearance, IL-7 expression was expressed throughout RAG2/ c and Ikaros fetal thymi in not detected by ISH in all epithelial cells within the thymic fated which there is a partial or complete block, respectively, in early The Journal of Immunology 65 thymopoiesis. Thus, hemopoietic-derived signals are not necessary generate B lineage-committed progeny in the bone marrow, CLPs for induction of IL-7 expression in epithelial cells of the early give rise to T cells in the thymic microenvironment (55, 56). How- thymic rudiment. However, it is not yet clear whether fetal TECs ever, distinct IL-7R-negative T lineage progenitors were recently are programmed to initiate IL-7 in a cell-autonomous fashion or described in the adult murine thymus (57, 58). Therefore, further whether exogenous signals are required. Given the general theme studies are needed to clarify the significance of IL-7 signaling for of inductive epithelial-mesenchymal interactions during embry- intrathymic hemopoietic progenitors. onic development and the fact that the thymic anlage is initially It is well established that lineage-committed DN thymocytes surrounded by neural crest mesenchyme (30, 49), it seems likely express IL-7Rs that transduce survival and differentiation signals that mesenchyme-derived signals play a role in inducing IL-7 as a result of IL-7 binding (11, 59). Differentiating DN precursors expression. migrate from the CMJ through the deep cortex to the subcapsular The localization pattern of IL-7-producing cells changes mark- region, where the DN3 to DN4 developmental transition occurs in edly throughout ontogeny. There is a widespread distribution of thymocytes that traverse the ␤-selection checkpoint (60). The im- IL-7-expressing TECs throughout the early fetal thymus. The ab- pact of IL-7 has been shown to be highly dose dependent with low sence of IL-7Rs on TEC progenitors suggests that IL-7 does not doses promoting and high doses inhibiting thymocyte development stimulate an autocrine signaling pathway. Rather, IL-7 appears to (61). Although IL-7-expressing TECs are sparsely distributed be readily available to hemopoietic precursors that enter the thy- throughout the cortex and subcapsular region of the adult thymus, mus via migration across the condensing mesenchymal capsule effective IL-7 signaling may nevertheless occur via several mech- (50). Because IL-7Rs are expressed on lymphoid progenitors that anisms. Trigueros et al. (11) demonstrated that IL-7 signals are Downloaded from emerge in the fetal liver at E11 to E12 (32–34), it is likely that T necessary for efficient progression of proliferating DN4 cells to the cell precursors are signaled by IL-7/IL-7R interactions upon en- DP stage. They further suggested that pre-TCR-mediated signaling countering the thymic microenvironment. The first wave of hemo- in DN4 thymocytes reduces the activation threshold, permitting an poietic precursors that migrates into the fetal thymus gives rise to effective response to low levels of IL-7 (11). A similar mechanism ␥␦ thymocytes that express an invariant TCR characterized by a was found to operate during B cell development in that pre-BCR- lack of junctional diversity (51, 52). IL-7R-mediated signals are

induced MAPK signals permit pre-B cells to respond to subopti- http://www.jimmunol.org/ ␥ required to initiate TCR gene rearrangement (13–15). Therefore, mal concentrations of IL-7 (62). Furthermore, IL-7 binds to extra- it seems likely that the prevalence of IL-7-expressing TECs in the cellular matrix (ECM) components such as fibronectin and heparan early fetal thymus is an important factor in establishing an appro- sulfate on TECs, resulting in activation of the surface integrin priate intrathymic milieu for promoting ␥␦ T cell development. In VLA-4, thereby enhancing thymocyte/epithelial interactions (29, contrast, IL-7 may play a redundant role in promoting in ␣␤ T cell 63, 64). Interestingly, DN thymocyte migration is directed in part development during fetal life because there is a relatively normal by interactions between surface integrins on thymocytes and ECM distribution, albeit reduced number, of each major thymocyte sub- components on TECs (65). Therefore, migrating DN thymocytes set in fetal IL-7R␣Ϫ/Ϫ mice, whereas thymocyte development is that bind to ECM components on cortical TECs are likely to en- arrested before the DN3 stage in the adult (16). by guest on September 30, 2021 counter low levels of IL-7 in the thymic cortex. In contrast to their widespread distribution in early ontogeny, a Development of the thymic epithelial microenvironment is con- striking dichotomy exists in the localization of IL-7-expressing sidered to occur in a stepwise manner dependent on signals from cells in cortical vs medullary compartments as the thymic micro- thymocytes at specific stages of maturation (66). The present study environment becomes specialized into functionally distinct zones demonstrates that distinct thymocyte subsets are responsible for of the late fetal and postnatal thymus. Earlier reports using RT- PCR showed that IL-7 is expressed by MHC class II-positive changes in the expression patterns of K5 and IL-7 in cortical TECs TECs, but the intrathymic localization of IL-7-expressing TECs of the postnatal thymus. We previously demonstrated that down- regulation of K5 in the thymic cortex depends on cross talk be- was not determined (12, 20, 53). Most notably, the medulla con- ϩ ϩ tains the highest concentration of IL-7-expressing cells in the adult tween K8 K5 TEC precursors and T lineage-committed thymo- thymus, whereas IL-7-expressing TECs are sparsely distributed in cytes (45, 67). Thus, K5 is down-regulated in the well-organized ⑀ the cortical and subcapsular regions. The high frequency of IL-7- cortex of RAG-1-deficient mice, whereas human CD3 transgenic ␥ Ϫ/Ϫ expressing cells in the medulla is not surprising given that IL-7Rs and RAG2/ c thymi that sustain earlier blocks in thymopoiesis ϩ Ϫ retain a primitive, poorly organized TEC population consisting of are up-regulated on positively selected CD4 CD8 and ϩ ϩ CD4ϪCD8ϩ thymocytes and that IL-7 drives expansion of SP K8 K5 TEC progenitors. We now show that in contrast to K5, medullary thymocytes (12). Furthermore, IL-7-mediated signals IL-7 continues to be expressed throughout the thymic epithelium promote the differentiation and maintain the viability of CD4- of RAG-1-deficient mice. Based on these data, we suggested that CD8ϩ thymocytes (39). The paucity of IL-7-expressing TECs in signals emanating from thymocytes that have advanced beyond the the cortex corresponds to the lack of IL-7R expression on DP DN3 stage of thymocyte development are required to induce IL-7 cortical thymocytes (54). In the absence of IL-7 signaling, nonse- down-regulation, or alternatively are required to stimulate selec- lected DP thymocytes down-regulate bcl-2 expression and undergo tive expansion of IL-7-negative precursors in the thymic cortex. apoptosis. This interpretation is consistent with the IL-7 expression pattern Ϫ Ϫ ϩ Although IL-7-expressing cells are relatively rare in the cortex, observed in RAG-2 / TCR␤ mice. Although IL-7-expressing IL-7 expression is pronounced in perivascular cells of the large cells were concentrated in the subcapsular region, the scarcity of vessels at the CMJ, where circulating hemopoietic precursors enter IL-7-expressing cells in thymic cortex was similar to the results the postnatal thymus (40, 41). Thus, it is likely that blood-borne obtained from ISH analysis of wild-type thymus. bone marrow progenitors encounter high concentrations of IL-7 as Finally, the presence of IL-7-expressing TECs throughout the Ϫ Ϫ they migrate out of the vascular system into the postnatal thymic RAG-1 / thymus, despite generation of a three-dimensional ep- microenvironment. Indeed, IL-7Rs are expressed on bone marrow- ithelial meshwork (66) consisting primarily of K8ϩK5Ϫ cells, in- derived common lymphoid progenitors (CLPs), which are clono- dicates that RAG-1Ϫ/Ϫ cortical TECs are not developmentally genic multipotent lymphoid-restricted precursors. Whereas CLPs equivalent to the wild-type cortical epithelial compartment. We 66 IL-7-EXPRESSING TECs

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