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* Science Park-Research Division, University of Texas M.D. Anderson Cancer Center, Smithville, TX 78957, and University of Texas Graduate School of Biomedical Sciences, Houston, TX 77225;
Department of Genetics, University of Georgia, Athens, GA 30602; and
Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain that is also a component of the thymic stromal lymphopoietin receptor, and the common cytokine receptor 
-chain (c)3 that is present in IL-2, -4, -9, -15, and -21 receptors (3). Signaling through the IL-7R initiates multiple signaling cascades, including activation of protein tyrosine kinases Jak1 and Jak3 that associate with the intracellular domains of the IL-7R and c chains, respectively. Activated Jak1 and Jak3 phosphorylate STAT1 and STAT5, resulting in altered gene expression patterns (4). IL-7-mediated signals modulate the expression of genes that affect thymocyte survival, proliferation, and differentiation. Disruption of IL-7 signaling in IL-7–/–, IL-7R–/–, and c–/– mice severely reduces thymic cellularity and impairs thymocyte development (5, 6, 7). This phenotype is due, in part, to reduced survival of CD4–CD8– double-negative (DN) thymocyte progenitors because introduction of a bcl-2 transgene or deletion of the death effector Bax rescues T cell development in IL-7R–/– and c–/– mice (8, 9, 10). IL-7 signaling also promotes the survival of DN thymocytes undergoing transition through the 
-selection checkpoint to the CD4+CD8+ double-positive (DP) stage (11) and proliferation of positively selected CD4+CD8– and CD4–CD8+ single-positive (SP) thymocytes (12). In addition to enhancing the survival and proliferation of -lineage thymocytes, IL-7 regulates TCR gene rearrangement by controlling locus accessibility, and is, therefore, essential for the development of 
T cells (13, 14, 15). Although recent studies suggest a less stringent requirement for IL-7 in promoting survival of fetal compared with adult thymocytes (13, 16), IL-7 expression has been reported in the early fetal thymus (17, 18). Intrathymic production of IL-7 is primarily a function of thymic epithelial cells (TECs) (19, 20). The TEC compartment is heterogeneous, consisting of subcapsular, cortical, and medullary subsets defined by morphological properties, antigenic 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 in the fetal and adult thymic microenvironment was not determined. Furthermore, the earliest developmental stage at which IL-7 is expressed in the thymic primordium has not been established. Given the functional significance of IL-7 signaling and the compartmentalization of thymocyte and epithelial subsets, we performed in situ hybridization (ISH) analyses to explore the emergence, distribution, and regulation of IL-7-expressing TECs during ontogeny and in postnatal mice. The data show striking differences in the localization and frequency of IL-7 expression in fetal and adult TECs. In addition, examination of IL-7 expression in the third pharyngeal pouch endoderm of wild-type and nude mice reveals IL-7 to be an early marker of TEC fate. Finally, differences in the thymocyte subsets that regulate IL-7 expression patterns demonstrate that cortical TECs in RAG-1–/– mice are developmentally and functionally immature compared with the wild-type cortical TEC compartment.
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Materials and Methods |
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C57BL/6J, RAG-1–/–, and 
/J mice were purchased from The Jackson Laboratory. RAG-2–/–/c–/– mice were purchased from Taconic Farms. Ikaros-null mice were the generous gift of K. Georgopoulos (Harvard Medical School, Charlestown, MA) (23). Embryos were obtained by setting up timed matings for 16 h and considering the morning of finding the vaginal plug as embryonic day 0.5 (E0.5). Embryos were fixed in Bouin’s solution for 3–5 h; washed overnight in 70% ethanol; dehydrated in series of 30, 50, 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 in situ hybridization, embryos were fixed in 4% paraformaldehyde/PBST overnight, washed twice in PBST, and dehydrated in series of 30, 50, 70, 90, and 100% methanol.
In situ hybridization
Paraffin section ISH was performed, as previously described (24), with the exception that the proteinase K digestion was omitted. The IL-7 riboprobe was obtained by RT-PCR on RNA from the cell line mouse plasmacytoma line J558 obtained from American Type Culture Collection (Manassas, VA). The PCR used the following oligos: 5' oligo, 5'-GCGACTGGATCCGACTACACCCACCTCCCGCAGACC-3'; 3' oligo, 5'-CGATCCGGATCCAAGATTCTTGGAGGTTGTTACTAC-3'.
The 5' oligo ends right before the ATG site, and the 3' oligo starts 30 bp downstream from the stop codon. After adding BamHI sites to each end, the 546-bp fragment was cloned into the blunted EcoRI site of BSII SK+ (Strategene). The 534-bp BamHI fragment was sequenced using T3 and T7 primers of BS.
The Gcm2 probe was generated by PCR amplification, as described (25). The Foxn1 probe was PCR amplified from E11.5 mouse cDNA using primers encompassing the eighth and ninth exons and 3' UTR (26). The IL-7, Foxn1, and Gcm2 riboprobes (sense and antisense) were labeled with digoxigenin during in vitro transcription. BM purple (Roche) was used as a chromagen for detection of the hybridized probe, and the sections were counterstained with nuclear fast red. Whole mount ISH was performed, as described (27).
Semiquantitative RT-PCR
Wild-type and nude E12 fetal thymic lobes were separately collected in TRIzol (Invitrogen Life Technologies) and homogenized using Micro Pellet Pestles (Nalge Nunc International). RNA was isolated, the genomic DNA was depleted with DNase I (Invitrogen Life Technologies), and. reverse transcription of total RNA to cDNA was performed using SuperScript II (Invitrogen Life Technologies), following manufacturer’s protocol. Equal amounts of cDNA from each strain were added to a final 20 µl 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 densities were measured and analyzed using the Molecular Analyst software (Bio-Rad, version 1.4.1). Primer sequences were: IL-7 (5'), 5'-ACT ACA CCC ACC TCC CGC A-3'; IL-7 (3'), 5'-TCT CAG TAG TCT CTT TAG G-3'; GADPH (5'), 5'-GTC TAC ATG TTC CAG TAT GAC TCC ACT CAC-3'; GADPH (3'), 5'-CAA TCT TGA GTG AGT TGT CAT ATT TCT CGT-3'; IL-7R (5'), 5'-GAC ATC AGA ATT CTT ACT GAT TGG-3'; IL-7R (3'), 5'-GGC GAG CTC GCC TTC GGG AAT GAA ACT CAC AT-3'; actin (5'), 5'-GTT TGA GAC CTT CAA CAC C-3'; actin (3'), 5'-GTG GCC ATC CCT GCT CGA AGT C-3'.
Immunohistology
Paraffin-embedded tissue sections were deparaffinized and rehydrated in 100–75% ethanol. OCT-embedded frozen tissue sections were air dried 30 min before acetone fixation. Sections were incubated overnight at 4°C with optimal dilutions of polyclonal anti-mouse keratin 5 (K5; Covance Research) and/or monoclonal anti-mouse c-kit (BD Pharmingen). Immunoreactivity to c-kit was enhanced by tyramide amplification (PerkinElmer Life Sciences). Controls included slides incubated with nonimmune rabbit-matched Ig or isotype-matched mouse Ig. For costaining, sections were incubated simultaneously with primary Abs from different species. Microscopic analysis was performed with an Olympus ProVis AX70 microscope (Olympus).
Thymic suspensions and FACS sorting
Thymocytes and TECs were released from E12.5 thymi by trypsin digestion, as previously described (28). Single cell suspensions were stained with allophycocyanin-conjugated anti-CD45 Ab (clone 30-F11) (BD Biosciences). Hemopoietic and stromal cells were isolated by sorting the Ab-stained cells for CD45+ (>98% purity) and CD45– (>94% purity) subsets, respectively, using a Corixa Elite flow cytometer.
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Results |
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IL-7 is a secreted cytokine that binds to MHC class II-positive TECs and fibroblasts in a heparin sulfate-dependent manner (29). To avoid detecting cells that bind, but do not synthesize IL-7, we evaluated IL-7 mRNA expression by ISH analysis. Although IL-7 expression was not detected in the pharyngeal region at E10.5 (data not shown), IL-7 message is expressed in the third pharyngeal pouch endoderm by E11.5. Fig. 1 shows that IL-7 expression is restricted to the epithelium of the third pouch and is not detected in other pharyngeal compartments. At E11.5, the parathyroid- and thymus-specific domains of the third pouch are contiguous within a common primordium that has not yet separated from the pharynx (30). A higher magnification view (Fig. 1B) shows that IL-7 expression is restricted to the ventral aspect of the third pouch, the domain that is fated to develop into thymus as opposed to parathyroid (31). Although IL-7 plays an essential role in thymocyte development, the early appearance of IL-7-expressing TECs during ontogeny suggested that it may function to stimulate TEC progenitors via an autocrine pathway. To explore this possibility, we assessed IL-7R expression on stromal as well as hemopoietic cells obtained from fetal thymi. Single cell suspensions of E12 thymi were stained with anti-CD45 and sorted to isolate CD45– stromal cells and CD45+ hemopoietic cells. Consistent with earlier reports showing that IL-R is expressed on fetal thymocyte progenitors (32, 33, 34), RT-PCR analysis of CD45+ cells revealed a strong IL-7R signal (Fig. 1C). In contrast, IL-7R expression was not detected in the CD45– compartment. Similar results were obtained with CD45+ and CD45– 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.
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Because Foxn1 is required for TEC differentiation and is expressed within a similar developmental time frame as IL-7, we considered that IL-7 might be a downstream target of Foxn1-mediated transcriptional regulation. To explore this possibility, we examined IL-7 expression in the rudimentary thymic remnant of early embryos from nude mice. Fig. 3, A and B, shows that IL-7 expression is readily detected by ISH analysis in the thymic primordium of E12 nude mice. Semiquantitative RT-PCR analysis confirmed the presence of IL-7 message in the E12 nude thymus anlage, although a stronger signal was observed in wild-type compared with nude fetal thymi (Fig. 3C). These results demonstrate that induction of IL-7 expression is independent of Foxn1-mediated regulation during thymic organogenesis. Furthermore, the presence of IL-7 message in the absence of functional Foxn1 establishes IL-7 as an early developmental marker of endodermal commitment to a thymic epithelial cell fate.
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As shown in Fig. 4, IL-7 is abundantly expressed throughout the E12.5 and E13.5 wild-type fetal thymus. By E15.5, there is a notable increase in thymic size and cellularity due to thymocyte and TEC proliferation. Thymic expansion is accompanied by a decreased frequency of IL-7-producing TECs particularly in the central region of the thymus. As development proceeds, the distribution of IL-7-expressing TECs becomes increasingly restricted such that by E16.5, IL-7-expressing TECs are found primarily toward the subcapsular region. In the neonatal thymus, IL-7 expression remains prominent in the subcapsular zone and is apparent at the junction between the cortex and the developing medullary regions (Fig. 5). The relatively low frequency of IL-7-expressing cells in the cortex could reflect down-regulation of IL-7 expression in cortical TECs or relocalization of IL-7-expressing TECs along with expansion of an IL-7-negative subset. The paucity of IL-7-expressing cells in the cortex is even more striking in 1-wk postnatal and adult thymi in which IL-7-expressing cells are concentrated in the corticomedullary junction (CMJ) and medulla. Expression of IL-7 in the medullary region is consistent with earlier reports showing that IL-7 promotes the proliferation and differentiation of SP thymocytes (12, 39). IL-7-expressing cells in the postnatal thymus are also associated with large blood vessels that are characteristically found in the perimedullary region at the CMJ (Fig. 5D). Hemopoietic 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 thymocyte progenitors enter the postnatal thymus, they encounter high concentrations of IL-7.
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We previously reported that hemopoietic-derived inductive signals are not required to establish initial patterning of fetal TEC subsets as determined by expression of distinct keratin species and antigenic profiles (42). To determine whether hemopoietic-derived signals are required to initiate IL-7 expression in the thymic rudiment, we examined IL-7-expressing TECs in two experimental models that sustain early blocks in T cell development. The Ikaros transcription factor plays an essential role in commitment of hemopoietic stem cells to the lymphoid lineage. Targeted deletion of the C-terminal region of the Ikaros gene results in a null phenotype 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-kit+CD25+ DN2 differentiation stage in mice that are deficient for both RAG-2 and the c receptor polypeptide chain (RAG2/c–/–) (43). As shown in Fig. 6A, fetal thymi from E13.5 RAG2/c–/– mice contain few c-kit-positive thymocyte progenitors, and such cells are undetectable in E13.5 Ikaros–/– thymi. Nevertheless, Fig. 6B shows that IL-7 is abundantly expressed in E13.5 fetal 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.
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Although thymocyte/TEC interactions are not required to specify phenotypic and functional characteristics of the thymic epithelial compartment during early ontogeny, thymocyte-derived signals are essential for proper organization and development of cortical and medullary TEC subsets during late fetal development and in the adult (42, 44, 45, 46). To determine whether thymocytes are also required to regulate IL-7 expression in the postnatal thymic epithelial compartment, we performed a comparative ISH analysis of IL-7 expression in adult thymi from wild-type, RAG-2/c–/–, and RAG-1–/– mice. In contrast to the developmental block at the DN2 stage in RAG-2/c–/– mice, thymocyte development is arrested at the DN3 stage in RAG-1–/– mice (43, 47).
Fig. 7 shows that, as noted above, IL-7-expressing TECs are predominantly localized in the medullary region of the wild-type adult thymus, whereas the majority of cortical TECs do not express detectable levels of IL-7 message. K5 is expressed by TECs in the medulla and at the corticomedullary junction, but not by cortical TECs (45). An analysis of K5 expression in a serial section confirmed the overlap in localization of IL-7 and K5 expression in the wild-type thymus. In striking contrast, IL-7-expressing TECs are present throughout the RAG-1–/– and RAG-2/c–/– thymi. The widespread IL-7 expression pattern in the adult RAG-2/c–/– thymus is consistent with the fact that the epithelial compartment in 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 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-1–/– 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 organize 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-1–/– cortex and reveal that TECs in the cortex of RAG-1–/– mice are developmentally and functionally distinct from wild-type cortical TECs.
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transgenic thymus
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-2–/– mice that express a TCR transgene. Introduction of a productively rearranged TCR transgene into RAG-2-deficient mice restores the ability of DN3 thymocytes to undergo -selection and differentiate to the DP stage (48). Although RAG-2–/–TCR+ 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-2–/–TCR+ mice (Fig. 8). In contrast to the consistent pattern of IL-7 expression found in the RAG-1–/– thymus, IL-7-expressing TECs are primarily concentrated in the subcapsular region of RAG-2–/–TCR+ mice. Furthermore, there is a notable decrease in the frequency of IL-7-expressing TECs in the expanded cortex, a phenotype that is similar to the paucity of IL-7-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 cortical TEC compartment.
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Discussion |
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IL-7 expression is initiated between E10.5 and E11.5 in the pharyngeal pouch endoderm of the common thymus/parathyroid primordium, specifically in the domain that is fated to develop into the thymic epithelial compartment. Thus, IL-7 expression can be considered as a functional marker of early TECs, and is likely to be expressed in the recently described K8+K5+MTS24+ progenitors that give rise to cortical and medullary epithelial compartments (37, 38). Despite its early appearance, IL-7 expression was not detected by ISH in all epithelial cells within the thymic fated domain. This may reflect maturation-dependent constraints on the level of IL-7 expression or the existence of distinct epithelial lineages. Further studies of precursor-progeny relationships and lineage analysis are necessary to distinguish these alternative explanations.
Although there are notable similarities in the temporal and spatial expression of IL-7 and Foxn1 during fetal thymic development, the presence of IL-7 mRNA in the nude thymic rudiment demonstrates that Foxn1 transcriptional activity is not required for IL-7 gene expression. The reduced IL-7 signal intensity in RT-PCR analysis of nude compared with wild-type fetal thymi suggests that Foxn1 may modulate the level of IL-7 expression. However, this interpretation is subject to the caveat that wild-type and nude thymi are not strictly comparable because thymic epithelial differentiation is arrested in nude mice and, therefore, the lower signal may be an indirect consequence of impaired TEC differentiation. Regardless, the Foxn1-independent expression of IL-7 in thymic fated epithelial cells of the common primordium identifies IL-7 as the earliest known marker of TEC fate in thymic ontogeny.
The early appearance of IL-7 message suggested that cross talk between epithelial and hemopoietic progenitors might not be required to initiate IL-7 expression in the fetal thymus. This premise was supported by ISH data showing that high levels of IL-7 are expressed throughout RAG2/c–/– and Ikaros–/– fetal thymi in which there is a partial or complete block, respectively, in early thymopoiesis. Thus, hemopoietic-derived signals are not necessary for induction of IL-7 expression in epithelial cells of the early thymic rudiment. However, it is not yet clear whether fetal TECs are programmed to initiate IL-7 in a cell-autonomous fashion or whether exogenous signals are required. Given the general theme of inductive epithelial-mesenchymal interactions during embryonic development and the fact that the thymic anlage is initially surrounded by neural crest mesenchyme (30, 49), it seems likely that mesenchyme-derived signals play a role in inducing IL-7 expression.
The localization pattern of IL-7-producing cells changes markedly throughout ontogeny. There is a widespread distribution of IL-7-expressing TECs throughout the early fetal thymus. The absence of IL-7Rs on TEC progenitors suggests that IL-7 does not stimulate an autocrine signaling pathway. Rather, IL-7 appears to be readily available to hemopoietic precursors that enter the thymus via migration across the condensing mesenchymal capsule (50). Because IL-7Rs are expressed on lymphoid progenitors that emerge in the fetal liver at E11 to E12 (32, 33, 34), it is likely that T cell precursors are signaled by IL-7/IL-7R interactions upon encountering the thymic microenvironment. The first wave of hemopoietic precursors that migrates into the fetal thymus gives rise to thymocytes that express an invariant TCR characterized by a lack of junctional diversity (51, 52). IL-7R-mediated signals are required to initiate TCR gene rearrangement (13, 14, 15). Therefore, it seems likely that the prevalence of IL-7-expressing TECs in the early fetal thymus is an important factor in establishing an appropriate intrathymic milieu for promoting T cell development. In contrast, IL-7 may play a redundant role in promoting in T cell development during fetal life because there is a relatively normal distribution, albeit reduced number, of each major thymocyte subset in fetal IL-7R–/– mice, whereas thymocyte development is arrested before the DN3 stage in the adult (16).
In contrast to their widespread distribution in early ontogeny, a striking dichotomy exists in the localization of IL-7-expressing cells in cortical vs medullary compartments as the thymic microenvironment becomes specialized into functionally distinct zones of the late fetal and postnatal thymus. Earlier reports using RT-PCR showed that IL-7 is expressed by MHC class II-positive TECs, but the intrathymic localization of IL-7-expressing TECs was not determined (12, 20, 53). Most notably, the medulla contains the highest concentration of IL-7-expressing cells in the adult thymus, whereas IL-7-expressing TECs are sparsely distributed in the cortical and subcapsular regions. The high frequency of IL-7-expressing cells in the medulla is not surprising given that IL-7Rs are up-regulated on positively selected CD4+CD8– and CD4–CD8+ thymocytes and that IL-7 drives expansion of SP medullary thymocytes (12). Furthermore, IL-7-mediated signals promote the differentiation and maintain the viability of CD4-CD8+ thymocytes (39). The paucity of IL-7-expressing TECs in the cortex corresponds to the lack of IL-7R expression on DP cortical thymocytes (54). In the absence of IL-7 signaling, nonselected DP thymocytes down-regulate bcl-2 expression and undergo apoptosis.
Although IL-7-expressing cells are relatively rare in the cortex, IL-7 expression is pronounced in perivascular cells of the large vessels at the CMJ, where circulating hemopoietic precursors enter the postnatal thymus (40, 41). Thus, it is likely that blood-borne bone marrow progenitors encounter high concentrations of IL-7 as they migrate out of the vascular system into the postnatal thymic microenvironment. Indeed, IL-7Rs are expressed on bone marrow-derived common lymphoid progenitors (CLPs), which are clonogenic multipotent lymphoid-restricted precursors. Whereas CLPs generate B lineage-committed progeny in the bone marrow, CLPs give rise to T cells in the thymic microenvironment (55, 56). However, distinct IL-7R-negative T lineage progenitors were recently described in the adult murine thymus (57, 58). Therefore, further studies are needed to clarify the significance of IL-7 signaling for intrathymic hemopoietic progenitors.
It is well established that lineage-committed DN thymocytes express IL-7Rs that transduce survival and differentiation signals as a result of IL-7 binding (11, 59). Differentiating DN precursors migrate from the CMJ through the deep cortex to the subcapsular region, where the DN3 to DN4 developmental transition occurs in thymocytes that traverse the -selection checkpoint (60). The impact of IL-7 has been shown to be highly dose dependent with low doses promoting and high doses inhibiting thymocyte development (61). Although IL-7-expressing TECs are sparsely distributed throughout the cortex and subcapsular region of the adult thymus, effective IL-7 signaling may nevertheless occur via several mechanisms. Trigueros et al. (11) demonstrated that IL-7 signals are necessary for efficient progression of proliferating DN4 cells to the DP stage. They further suggested that pre-TCR-mediated signaling in DN4 thymocytes reduces the activation threshold, permitting an effective response to low levels of IL-7 (11). A similar mechanism was found to operate during B cell development in that pre-BCR-induced MAPK signals permit pre-B cells to respond to suboptimal concentrations of IL-7 (62). Furthermore, IL-7 binds to extracellular matrix (ECM) components such as fibronectin and heparan sulfate on TECs, resulting in activation of the surface integrin VLA-4, thereby enhancing thymocyte/epithelial interactions (29, 63, 64). Interestingly, DN thymocyte migration is directed in part by interactions between surface integrins on thymocytes and ECM components on TECs (65). Therefore, migrating DN thymocytes that bind to ECM components on cortical TECs are likely to encounter low levels of IL-7 in the thymic cortex.
Development of the thymic epithelial microenvironment is considered to occur in a stepwise manner dependent on signals from thymocytes at specific stages of maturation (66). The present study demonstrates that distinct thymocyte subsets are responsible for changes in the expression patterns of K5 and IL-7 in cortical TECs of the postnatal thymus. We previously demonstrated that down-regulation of K5 in the thymic cortex depends on cross talk between K8+K5+ TEC precursors and T lineage-committed thymocytes (45, 67). Thus, K5 is down-regulated in the well-organized cortex of RAG-1-deficient mice, whereas human CD3
transgenic and RAG2/c–/– thymi that sustain earlier blocks in thymopoiesis retain a primitive, poorly organized TEC population consisting of K8+K5+ TEC progenitors. We now show that in contrast to K5, IL-7 continues to be expressed throughout the thymic epithelium of RAG-1-deficient mice. Based on these data, we suggested that signals emanating from thymocytes that have advanced beyond the DN3 stage of thymocyte development are required to induce IL-7 down-regulation, or alternatively are required to stimulate selective expansion of IL-7-negative precursors in the thymic cortex. This interpretation is consistent with the IL-7 expression pattern observed in RAG-2–/–TCR+ mice. Although IL-7-expressing cells were concentrated in the subcapsular region, the scarcity of IL-7-expressing cells in thymic cortex was similar to the results obtained from ISH analysis of wild-type thymus.
Finally, the presence of IL-7-expressing TECs throughout the RAG-1–/– thymus, despite generation of a three-dimensional epithelial meshwork (66) consisting primarily of K8+K5– cells, indicates that RAG-1–/– cortical TECs are not developmentally equivalent to the wild-type cortical epithelial compartment. We speculate that RAG-1–/– cortical TECs are arrested at an immature stage of development due to the absence of appropriate inductive signals generated by thymocytes that have traversed the DN3 to DN4 checkpoint.
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Acknowledgments |
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Footnotes |
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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 Institute on Environmental Health Sciences Center Grant ES07784 (to E.R.), and National Institutes of Health Grant AI055001 (to N.M.). 
2 Address correspondence and reprint requests to Dr. Ellen Richie, P.O. Box 389, Science Park-Research Division, Smithville, TX 78957. E-mail address: erichie{at}odin.mdacc.tmc.edu
3 Abbreviations used in this paper: c, common -chain; CLP, common lymphoid progenitor; CMJ, corticomedullary junction; DN, double negative; DP, double positive; E, embryonic day; ECM, extracellular matrix; ISH, in situ hybridization; K5, keratin 5; SP, single positive; TEC, thymic epithelial cell.
Received for publication March 9, 2004. Accepted for publication October 18, 2004.
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C. Soza-Ried, C. C. Bleul, M. Schorpp, and T. Boehm Maintenance of Thymic Epithelial Phenotype Requires Extrinsic Signals in Mouse and Zebrafish J. Immunol., October 15, 2008; 181(8): 5272 - 5277. [Abstract] [Full Text] [PDF]
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 I. Popa, I. Zubkova, M. Medvedovic, T. Romantseva, H. Mostowski, R. Boyd, and M. Zaitseva Regeneration of the adult thymus is preceded by the expansion of K5+K8+ epithelial cell progenitors and by increased expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma Int. Immunol., November 1, 2007; 19(11): 1249 - 1260. [Abstract] [Full Text] [PDF]
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L. Gautreau, M.-L. Arcangeli, V. Pasqualetto, A.-M. Joret, C. Garcia-Cordier, J. Megret, E. Schneider, and S. Ezine Identification of an IL-7-Dependent Pre-T Committed Population in the Spleen J. Immunol., September 1, 2007; 179(5): 2925 - 2935. [Abstract] [Full Text] [PDF]
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M. Itoi, N. Tsukamoto, H. Yoshida, and T. Amagai Mesenchymal cells are required for functional development of thymic epithelial cells Int. Immunol., August 1, 2007; 19(8): 953 - 964. [Abstract] [Full Text] [PDF]
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L. Zeng, S. L. Dalheimer, and T. M. Yankee Gads-/- Mice Reveal Functionally Distinct Subsets of TCRbeta+ CD4-CD8- Double-Negative Thymocytes J. Immunol., July 15, 2007; 179(2): 1013 - 1021. [Abstract] [Full Text] [PDF]
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M. Itoi, N. Tsukamoto, and T. Amagai Expression of Dll4 and CCL25 in Foxn1-negative epithelial cells in the post-natal thymus Int. Immunol., February 1, 2007; 19(2): 127 - 132. [Abstract] [Full Text] [PDF]
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 W. E. Jenkinson, S. W. Rossi, S. M. Parnell, E. J. Jenkinson, and G. Anderson PDGFR{alpha}-expressing mesenchyme regulates thymus growth and the availability of intrathymic niches Blood, February 1, 2007; 109(3): 954 - 960. [Abstract] [Full Text] [PDF]
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 T. Yokota, J. Huang, M. Tavian, Y. Nagai, J. Hirose, J.-C. Zuniga-Pflucker, B. Peault, and P. W. Kincade Tracing the first waves of lymphopoiesis in mice Development, May 15, 2006; 133(10): 2041 - 2051. [Abstract] [Full Text] [PDF]
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 Q. Yu, J.-H. Park, L. L. Doan, B. Erman, L. Feigenbaum, and A. Singer Cytokine signal transduction is suppressed in preselection double-positive thymocytes and restored by positive selection J. Exp. Med., January 23, 2006; 203(1): 165 - 175. [Abstract] [Full Text] [PDF]
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I. Zubkova, H. Mostowski, and M. Zaitseva Up-Regulation of IL-7, Stromal-Derived Factor-1{alpha}, Thymus-Expressed Chemokine, and Secondary Lymphoid Tissue Chemokine Gene Expression in the Stromal Cells in Response to Thymocyte Depletion: Implication for Thymus Reconstitution J. Immunol., August 15, 2005; 175(4): 2321 - 2330. [Abstract] [Full Text] [PDF]
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