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Thymus Medulla Formation and Central Tolerance Are Restored in IKK^–/– Mice That Express an IKK Transgene in Keratin 5⁺ Thymic Epithelial Cells¹

Department of Carcinogenesis, Science Park Research Division, University of Texas M. D. Anderson Cancer Center, Smithville, TX 78957

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-B pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IB kinase (IKK), a required intermediate in the noncanonical NF-B signaling pathway. Introduction of an IKK transgene driven by a K5 promoter restores the K5⁺K14⁺ mTEC subset in IKK^–/– mice. Unexpectedly, the K5-IKK transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKK transgene ameliorates autoimmune symptoms in IKK^–/– mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-B signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The thymus provides a unique microenvironment that is responsible for the development of T cells that respond to foreign peptides presented by self-MHC molecules, yet are tolerant of self-peptide-MHC complexes. Interactions between thymocytes and thymic epithelial cells (TECs)³ are required to shape the TCR repertoire during thymocyte differentiation (1, 2). Cortical TECs (cTECs) display peptide-MHC complexes that induce positive selection of CD4⁺CD8⁺ double positive thymocytes expressing TCRs with relatively low peptide binding affinity. Signaling pathways activated during positive selection promote continued differentiation of double positive thymocytes to the CD4⁺CD8^– or CD4^–CD8⁺ single positive stage and initiate thymocyte migration into the medulla. Medullary TECs (mTECs) play a key role in negatively selecting potentially autoreactive thymocytes. One primary mechanism for establishing central tolerance to autoantigens is the induction of apoptosis in thymocytes that express high affinity TCRs for peptide-MHC complexes on the surface of mTECs. Medullary TECs are also required for the generation of CD4⁺CD25⁺ T regulatory cells (Tregs) and NKT cells, both of which actively repress self-reactive T cells (3, 4).

The requirement for mTECs in generating central tolerance is primarily a function of their unique ability to express genes encoding a wide range of Ags many of which were previously considered to be expressed only in peripheral tissues (reviewed in Ref. 5). This phenomenon, referred to as promiscuous gene expression is regulated in part by the autoimmune regulator (AIRE) gene, which is preferentially expressed in mTECs. The role of AIRE gene in establishing central tolerance is illustrated by a rare syndrome, termed APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy), in which individuals with AIRE mutations develop severe, multiorgan autoimmune disease (6). Animal models of AIRE deficiency have verified that self-reactive thymocytes persist due to defective clonal deletion, although additional undefined factors regulate tissue-restricted Ag (TRA) expression because certain TRAs are expressed even in the absence of AIRE (7, 8). In addition to direct presentation, mTECs supply self-peptides to medullary dendritic cells (DCs), which process and cross-present them to augment deletion of self-reactive T cells (9). DCs and mTECs both express CD40, CD80, and CD86 costimulatory molecules that enhance negative selection as well as promote development of Tregs (10, 11, 12, 13). Thus, mTECs contribute directly and indirectly to establishing central tolerance and averting the development of autoimmunity.

The medullary compartment contains phenotypically diverse epithelial cells that can be categorized into two major subsets. One mTEC subset displays high levels of surface MHC class II molecules, binds the lectin Ulex europaeus agglutinin-1 (UEA-1), expresses keratin 8 (K8) but not K5, and has a compact, globular appearance (14, 15, 16). A distinct mTEC subset expresses the nonpolymorphic MHC class II molecule I-O, fails to bind UEA-1, expresses K5 but not K8, and has a more stellate morphology (15, 16). Although both medullary subsets express CD80, a positive correlation has been found between the level of UEA-1 binding sites and CD80 expression (8). Interestingly, AIRE and TRAs are expressed at higher levels in UEA-1^high or CD80^high cells prompting the suggestion that these cells represent a more advanced stage of mTEC differentiation (8, 17). However, a direct precursor-progeny relationship between phenotypically or functionally distinct mTEC subsets has not been demonstrated.

The development of both mTEC subsets as well as thymic DCs depends on activation of the NF-B signaling pathway, specifically the noncanonical, alternative pathway that culminates in RelB activation (18). Ligand engagement of certain receptors in the TNFR family such as lymphotoxin receptor (LTR) activates NF-B-inducing kinase (NIK), which phosphorylates homodimers of the downstream IB kinase (IKK). Activated IKK in turn phosphorylates the C-terminal region of NF-B2 (p100) leading to ubiquitin-dependent degradation and release of the N-terminal polypeptide, p52. The formation of RelB/p52 heterodimers permits shuttling of RelB from the cytoplasm into the nucleus where it functions as a transcriptional regulator (18). A range of medullary defects occurs in mice that are deficient in various components of the alternative NF-B activation pathway. Targeted disruption of the LTR gene results in disorganized medullary regions that contain reduced numbers of both major mTECs subsets (19). Mice that have a naturally occurring mutation in NIK (NIK^aly/aly) not only fail to develop lymph nodes and Peyer’s patches, but also have severe defects in medulla formation, including a deficiency of UEA-1 binding mTECs and reduced AIRE expression (20). A similar medullary phenotype is found in mice that are deficient in TNFR-associated factor-6 (21). Furthermore, DCs as well as UEA-1 binding TECs are absent in RelB-deficient mice (22, 23). Interestingly, these mutant strains also develop severe inflammation and autoimmune disease, indicating a breakdown in the establishment of central tolerance.

Although there is ample experimental evidence that RelB activation is essential for mTEC development and medullary organization, it is not clear whether activation of the alternative NF-B pathway is required for the differentiation and function of both major mTEC subsets. To address this issue we analyzed mice in which a targeted mutation in IKK prevents noncanonical NF-B activation (24, 25) and asked whether restoration of IKK expression exclusively in the K5⁺ mTEC subset is sufficient to restore the generation of UEA-1 binding cells and prevent development of autoimmune disease. Medulla formation and mTEC development are severely impaired in IKK^–/– mice, and autoimmune-like pathology develops when IKK^–/– fetal thymuses are transferred into athymic recipients. Introduction of an IKK transgene regulated by a K5 promoter rescues development of the K5^–UEA-1^high as well as the K5⁺UEA-1^neg mTEC and prevents the development of autoimmunity. This outcome suggests that 1) medulla formation and central tolerance depend on activating the alternative NF-B signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

NCr^nu/nu and BALB/c^nu/nu mice were purchased from National Cancer Institute. IKK^–/– mice were generated and genotyped as previously described (24). The IKK^–/– mice were originally on a C57BL6/129Sv background and backcrossed onto C57BL6/J for more than five generations. 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.

To produce the K5-IKK transgenic line, a human IKK cDNA fragment tagged with hemagglutinin was generated by PCR from a DNA template of an IKK-expressing vector (26). The IKK cDNA PCR fragment was inserted into the pGEM-T vector (Promega) sequening, and subsequently subcloned into the NotI and SnaB1 sites of the BK5 vector containing a bovine K5 promoter as previously described (27). The construct DNA was linearized by XhoI digestion and used to generate K5-IKK transgenic mice in the transgenic core located at the Science Park Research Division of the University of Texas M. D. Anderson Cancer Center (Smithville, TX). The PCR primers 5'-AAAGTGTGGGCTGAAGCAGTG-3' and 5'-GCCCAACAACTTGCTCAAATG-3' (1273–1819 bp) generate a 546 bp DNA fragment for genotyping. IKK^+/– and K5-IKK transgenic mice were crossed to obtain K5-IKK/IKK^–/– mice that were identified by genotyping. Unless stated otherwise, age-matched embryonic day 18.5 or newborn mice were used for all experiments.

Immunohistochemistry

Serial sections (5 µm) from OCT-embedded frozen tissue were air dried and fixed in cold acetone for 2 min at room temperature. After washing in HBSS, they are blocked for 15 min in HBSS containing 1% BSA. The slides were incubated at 4°C with optimal dilutions of primary Abs. The following Abs and reagents were used: polyclonal anti-mouse K5 and K14 (Covance), Troma-1 mAb (rat anti-K8; Developmental Studies Hybridoma Bank) and hamster anti-mouse CD11c (clone HL3; BD Biosciences). Biotinylated and FITC-conjugated UEA-1 were obtained from Vector Laboratories. Control slides were incubated with nonimmune serum or isotype-matched Ig. After washing, sections were incubated 30 min with directly conjugated secondary reagents. For CD11c, immunoreactivity was enhanced by tyramide amplification (PerkinElmer Life Sciences). Microscopic analysis was performed with an Olympus ProVis AX70 microscope.

Thymic stromal cell suspensions

Single-cell suspensions from thymuses of 4- to 6-wk-old K5-IKK mice were prepared as described by Gray et al. (28) with minor modifications. Thymuses were finely minced and gently stirred in a beaker in cold RPMI 1640 containing 1–5% FBS. Passing the suspension through a 70-µm cell strainer (BD Falcon) depleted thymocytes. Fresh medium was added to the stromal fragments recovered from the strainer and the process was repeated three or four times. Stromal fragments were washed in Ca²⁺ and Mg²⁺ free PBS and digested with 0.25% trypsin (Sigma-Aldrich) in 0.02% EDTA for 45 min at 37°C during which they were dispersed several times by pipetting. Digestion was stopped by washing in RPMI 1640 containing 5% FBS. The resulting suspension was further depleted of thymocytes by incubation with anti-mouse CD45 bound magnetic beads (Dynal Biotech) and depleting the bound cells with a magnet.

Flow cytometric analysis and sorting

Flow cytometric analysis and sorting was performed Beckman Coulter Altra flow cytometer. The following mAbs were purchased from BD Biosciences: anti-CD4 (RM4-5) conjugated with PE, anti-CD8 (53-6.7) conjugated with FITC, FITC-conjugated anti-CD45.2, biotinylated anti-CD62 ligand (MEL-14), and biotinylated anti-CD44 (IM7). Monoclonal Ab to Foxp3 (clone FJK-16s) was purchased from eBioscience. Thymocytes in HBSS containing 1% BSA and 0.1% sodium azide were incubated with directly conjugated or biotinylated Abs on ice for 30 min followed by three washes. Biotinylated Abs were detected with streptavidin-allophycocyanin. The cells were fixed in 1% paraformaldehyde and stored at 4°C until analysis. For sorting thymus stromal cells, the CD45-depleted cell suspensions were incubated with allophycocyanin-conjugated anti-CD45 (clone 30-F11) and FITC-conjugated UEA-1 in the presence of 2 µg/ml propidium iodide to exclude dead cells. CD45 and propidium iodide-positive cells were excluded by electronic gating and UEA^neg-low, UEA^int, and UEA^high cells were collected using the sort gates (see Fig. 5).

View larger version (22K): [in this window] [in a new window]   FIGURE 5. K5 and TRA expression in K5-IKK thymic stromal cells isolated by differential UEA-1 binding. A, RT-PCR analysis of IKK transgene expression in FACS sorted CD45– thymic stromal cells and CD45+ hemopoietic cells from 4-wk-old K5-IKK transgenic mice. A representative result from three individual animals is shown. B, CD45– thymic stromal cells from 4-wk-old K5-IKK transgenic thymuses were pooled and incubated with FITC-conjugated UEA-1 and FACS sorted using the indicated gates to obtain UEA-1neg-low (59% of stromal cells), UEA-1int (26% of stromal cells), and UEA-1high (14% of stromal cells) populations. C, RT-PCR analysis of isolated UEA-1neg-low, UEA-1int, and UEA-1high cells for IKK transgene, K5, AIRE, and TRA expression. A representative result from three similar experiments is shown.

Fetal thymic lobes from embryonic day 15.5 IKK^–/–, K5-IKK/IKK^–/–, and their corresponding wild-type littermates were placed on Nucleopore filters (Whatman) and cultured for 4 days in complete RPMI 1640 containing 10% FBS and 1.35 mM 2-deoxyguanosine. The thymocyte-depleted lobes from IKK^–/– and K5-IKK/IKK^–/– mice were grafted under the renal capsule of NCr^nu/nu and BALB/c^nu/nu mice, respectively, as previously described (29). After 6–8 wk, the grafted thymuses were recovered for immunohistochemical analysis and peripheral lymph nodes were obtained for analysis of T cell development by flow cytometric analysis. Peripheral lymph node cells obtained from K5-IKK/IKK^–/– thymus graft recipients were electronically gated for CD45.2 and then analyzed for activation phenotype.

Pathology

Antinuclear Abs (ANA) were detected by using mouse ANA ELISA kit (Alpha Diagnostic International). To examine tissues for lymphocytic infiltration, formalin-fixed slides were stained with H&E.

Semiquantitative RT-PCR analysis

RNA was prepared using the Absolutely RNA Microprep kit (Stratagene) from thymic stromal cell preparations depleted of CD45⁺ hemopoietic cells by anti-CD45 bound magnetic beads or from isolated stromal subsets obtained by cell sorting. Equal amounts of RNA were subjected to oligo(dT)-primed reverse transcription using SuperScript II (Invitrogen Life Technologies), following the manufacturer’s protocol. Equal amounts of cDNA from each sample were added to a final 25-µl PCR mix using the Qiagen TaqPCR kit. The following PCR conditions were used: initial denaturation of 94°C 5 min, followed by either 40 cycles (transgene), 35 cycles (promiscuously expressed genes and AIRE), or 33 cycles (RelB, K5, chemokines, and actin) of 94°C for 0.5–1 min, 55–60°C for 0.5–1 min, and 72°C for 1 min, and final extension of 72°C for 5 min. The following primer sequences were used: AIRE, (forward) 5'-GAC CTA AAC CAG TCC CGG AA-3', (reverse) 5'-ATC CCT TCC ACG GCC CCT-3'; salivary protein 1, (forward) 5'-GGC TCT GAA ACT CAG GCA GA-3', (reverse) 5'-TGC AAA CTC ATC CAC GTT GT-3'; C-reactive protein, (forward) 5'-CCA TGG AGA AGC TAC TCT G-3', (reverse) 5'-CCC AAG ATG ATG CTT GC-3'; fatty acid binding protein, (forward) 5'-AGA CGG AAC GGA GCT CAC-3', (reverse) 5'-GCT CTT CAG CGT TGC TCC-3'; insulin, (forward) 5'-AGA CCA TCA GCA AGC AGG TC-3', (reverse) 5'-CTG GTG CAG CAC TGA TCC AC-3'; actin, (forward) 5'-GTT TGA GAC CTT CAA CAC C-3', (reverse) 5'-GTG GCC ATC CCT GCT CGA AGT C-3'; CCL19, (forward) 5'-GCT AAT GAT GCG GAA GAC TG-3', (reverse) ACT CAC ATC GAC TCT CTA GG-3'; CCL21, (forward) 5'-GCT GCC TTA AGT ACA GCC AG-3', (reverse) 5'-GTG TCT GTT CAG TTC TCT TGC-3'; RelB, (forward) 5'-CAA GAA GTC CAC CAA CAC ATC-3', (reverse) 5'-GGA AGT GGT CCA AGA ACA CTG-3'; IKK transgene, (forward) 5'-GCC ATG TAC CCA TAC GAT GTT CC-3', (reverse) 5'-GCT CCA ATA ATC AAC AGT GGC TG-3'; and K5, (forward) 5'-GAT GCT GCC TAC ATG AAC AAG-3', (reverse) 5'-TCC AGC TCT GTC AGC TTG TT-3'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IKK is required for development of mTECs and DCs

IKK^–/– mutant mice die shortly after birth and display numerous developmental defects including failure of limb development, abnormal skeletal patterning and hyperplasia of the suprabasal epidermal layer resulting from a block in keratinocyte differentiation (24, 25, 30). In addition, we invariably observed thymus hypoplasia in newborn IKK^–/– mice (Fig. 1A). Despite a marked reduction in cellularity, (3.3 ± 0.3 x 10⁶ IKK^–/– compared with 8.9 ± 0.4 x 10⁶ IKK^+/+ thymocytes), the relative percentage of thymocyte subsets defined by CD4 and CD8 expression was not altered in the absence of IKK (data not shown). However, inspection of H&E stained sections revealed an absence of distinct medullary regions in the IKK^–/– thymus (Fig. 1A). Immunohistochemical staining with Abs to mouse K8 and K5 showed a well organized network of K8⁺K5^– cTECs in the IKK^–/– newborn thymus that appeared comparable to cTECs in IKK^+/+ littermate controls. In contrast, mTEC development was severely impaired in the IKK^–/– thymus as shown in Fig. 1B. There was a marked reduction in K8^–K5⁺ mTECs, and those that were present appeared to be condensed into atypical, compact clusters. In addition, there was an almost complete absence of the UEA-1 binding mTEC subset. These findings are consistent with a recent report describing the medullary phenotype in an independently derived IKK^–/– line (31). Overall, the TEC phenotype in IKK^–/– mice is similar to that reported in LTR^–/–, NIK^aly/aly, and RelB^–/– mice (19, 20, 22). However, in contrast to LTR^–/– and NIK^aly/aly mice, there is a marked depletion of thymic DCs in IKK^–/– and RelB^–/– mice.

View larger version (61K): [in this window] [in a new window]   FIGURE 1. IKK is required for normal development of mTECs and DCs. A, H&E stained frozen sections (5 µm) revealed thymus hypoplasia and absence of medullary regions in the IKK–/– thymus compared with wild-type littermate thymus at original magnification x40 (top) and at magnification x100 (bottom). B, Serial frozen sections were stained with Abs to K8, K5, and CD11c and fluorochrome-conjugated second step reagents or biotinylated UEA-1 and streptavidin-FITC as indicated. Images shown are representative of five different thymuses of each genotype at original magnification of x100.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Reduced AIRE, TRA, and chemokine expression in IKK–/– thymic stromal cells. Semiquantitative RT-PCR analysis was performed using RNA obtained from CD45-depleted stromal cells from IKK–/– and IKK+/+ newborn thymuses. Three-fold serial dilutions of cDNA were used. -actin served as a loading control for each sample. A representative RT-PCR analysis from three experiments is shown.

Impaired mTEC development in IKK^–/– embryos is a cell autonomous defect

Because thymocyte-derived signals are required for development and maintenance of the medullary compartment (34, 35), the mTEC deficiencies in IKK^–/– mice could be an indirect consequence of defective cross-talk between thymocytes and mTEC progenitors. To explore this issue, embryonic day 15.5 fetal thymus lobes from IKK^–/– or IKK^+/+ littermates were depleted of thymocytes by 2-deoxyguanosine treatment and transplanted under the kidney capsule of athymic nude (NCr^nu/nu) mice. IKK sufficient hemopoietic precursors in the recipient bone marrow migrate to and differentiate within the stromal microenvironment of the thymus graft. As shown in Fig. 3, the IKK^+/+ thymus grafts recovered 8 wk after transplantation contained well-organized medullary regions with an abundant network of stellate K5⁺ mTECs that coexpress K14, a typical K5 binding partner. The presence of UEA-1 binding mTECs intermingled among the K5⁺K14⁺ cells was further evidence of normal medullary epithelial development in the IKK^+/+ thymus graft. In striking contrast, the transplanted IKK^–/– thymus contained only small medullary-like regions that contained few K5⁺K14⁺ mTECs. UEA-1 binding mTECs were undetectable. These data demonstrate that the deficiency of mTEC development in IKK^–/– mice is an epithelial cell autonomous defect. Conversely, DCs were present in the IKK^–/– thymus grafts suggesting that the absence of IKK in hemopoietic precursors was a major factor responsible for the DC deficiency in IKK^–/– mice.

View larger version (92K): [in this window] [in a new window]   FIGURE 3. Impaired mTEC development in IKK–/– mice is a cell autonomous defect. Embryonic day 15.5 thymic lobes from IKK–/– and IKK+/+ embryos were incubated for 4 days in the presence of 2-deoxyguanosine and transplanted under the kidney capsule of athymic recipients. Eight weeks later, the transplanted thymuses were recovered and stained with H&E, Abs to K5, K8, K14, and CD11c, and the lectin UEA-1 as indicated. The cortex (C) and medulla (M) are indicated. IKK+/+ thymus grafts contained well-organized medullary regions with K5+ and UEA-1 binding mTEC subsets, whereas IKK–/– thymus grafts contained few K5+ and undetectable UEA-1 binding mTECs. The original magnification was x100. The images shown are representative of four different thymus grafts.

IKK is required for development of self-tolerance

Recent studies have shown that mTECs play a critical role in establishing central tolerance by promiscuous expression of TRAs that negatively select autoreactive T cell clones (7, 8, 17, 36). Consistent with this notion, impaired negative selection and autoimmunity are associated with experimental models in which mTEC development is defective due to a block in the nonclassical NF-B signaling pathway (e.g., LTR^–/–, NIK^aly/aly, RelB^–/– mice) (19, 20, 22, 23). Thus, it was not surprising to find activated peripheral T cells and autoimmune manifestations in NCr^nu/nu recipients of IKK^–/– fetal thymus grafts. As shown in Fig. 4A both IKK^+/+ and IKK^–/– fetal thymus grafts support the development of CD4⁺ and CD8⁺ lymph node T cells. However, CD4⁺ peripheral T cells from recipients of IKK^–/–, but not IKK^+/+, grafts displayed an activated phenotype based on increased CD44 and decreased CD62L expression (Fig. 4B). The IKK^–/– thymus grafts also appeared to be inefficient in supporting the generation of peripheral Tregs because expression levels of Foxp3 were reduced in isolated CD4⁺CD25⁺ splenic T cells from recipients of IKK^–/– compared with wild-type thymus grafts (compare with Fig. 7D). Furthermore, there was a low frequency of Foxp3 expressing cells detected by immunohistochemistry in IKK^–/– thymus grafts (data not shown). IKK^–/– graft recipients produced high levels of serum ANA (see Fig. 7C) and developed prominent perivascular lymphocytic infiltrates in liver and pancreas (Fig. 4C). These results suggest that impaired mTEC development in the IKK^–/– thymus results in the generation of activated peripheral T cells, reduced Tregs and a profound deficit in the establishment of central tolerance.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Activated peripheral T cells and impaired central tolerance in athymic recipients of IKK–/– fetal thymus grafts. A, Flow cytometric analysis of CD4+ and CD8+ T cells in peripheral lymph nodes from recipients of IKK+/+ or IKK–/– fetal thymus lobes. B, Electronic gates were set on CD4+ lymph node T cells to determine the frequency of CD44high and CD62Llow cells in recipients of IKK–/– () or IKK+/+ () fetal thymus grafts. C, H&E stained formalin-fixed sections of liver and pancreas from recipients of IKK–/– and IKK+/+ fetal thymus grafts. Arrows indicate lymphoid cell infiltrates. The results shown are representative of four experiments.

View larger version (52K): [in this window] [in a new window]   FIGURE 7. Central tolerance is restored in IKK–/– mice that express a K5-IKK transgene. A, Embryonic day 15.5 thymic lobes from K5-IKK/IKK–/– and wild-type embryos were transplanted under the kidney capsule of athymic recipients. Six weeks later the transplanted thymuses were recovered and stained as indicated. Staining for TEC subsets in the K5-IKK/IKK–/– transplanted thymus is shown; TEC development in the wild-type transplanted thymus was comparable to the results in Fig. 3 (data not shown). The experiment was repeated twice with the same results. B, Flow cytometric analysis of CD4+ lymph node T cells from athymic recipients that were grafted with either K5-IKK/IKK–/– () or wild-type () embryonic day 15.5 thymus lobes. Electronic gates were set on CD4+ lymph node cells to analyze the frequency of CD44high and CD62Llow CD4+ T cells. The values shown are the average of three different experiments. C, Serum ANA were detected by ELISA in serum from athymic recipients grafted with wild-type (), IKK–/– (), or K5-IKK/IKK–/– () embryonic day 15.5 thymus lobes. Absorbance is directly proportional to the amount of ANA present in each sample. Results are expressed as mean ± SD of values obtained in two independent experiments. D, RT-PCR analysis of Foxp3 expression in FACS sorted CD4+CD25+ splenocytes. E, H&E staining of formalin-fixed liver and pancreas sections from athymic recipients of K5-IKK/IKK–/– thymic lobes. Note the absence of lymphoid cell infiltrates in comparison to the marked infiltration observed in recipients of IKK–/– thymus lobes (compare with Fig. 4C).

A K5 promoter-driven IKK transgene rescues medulla formation in IKK^–/– mice

It has been suggested that increased expression of CD80, MHC class II, and UEA-1 binding molecules correlates with increased mTEC maturation, although a direct lineage relationship between phenotypically distinct mTEC subsets has not been established (17). Moreover, it is not clear whether maturation and/or function of mTECs depends on activating the alternative NF-B pathway in both the K5⁺ and UEA-1 binding mTEC subsets, or alternatively, whether RelB activation in the K5⁺ mTEC subset is sufficient to directly or indirectly induce development of UEA-1 binding mTECs. To address this issue, we asked whether restoring IKK expression exclusively in the K5⁺ mTEC subset would rescue generation of UEA-1 binding mTECs. To this end, we generated a novel transgenic mouse model in which expression of a human IKK transgene is expressed under the control of a K5 promoter. The K5-IKK transgenic mice are healthy and fertile. Fig. 5A shows that transgene expression in the thymus is restricted to CD45-negative thymic stromal cells and is not detected by RT-PCR analysis in CD45-positive hemopoietic cells. CD45-negative thymic stromal cells from K5-IKK transgenic mice were incubated with FITC-conjugated UEA-1 and FACS sorted into UEA-1^neg-low, UEA-1^int, and UEA-1^high populations (Fig. 5B). Semiquantitative RT-PCR analysis revealed that endogenous K5 as well as the IKK transgene were highly expressed in the UEA-1^neg-low population (Fig. 5C). Importantly, neither K5 nor the IKK transgene were expressed at detectable levels in UEA-1^high cells. Consistent with a previous report (8), AIRE and AIRE-dependent TRAs salivary protein 1 and fatty acid binding protein were expressed at highest levels in UEA-1^high TECs. In contrast, C-reactive protein, an AIRE-independent TRA was expressed at similar levels in both the UEA-1^neg-low and UEA-1^high cells. These data are consistent with recent detailed analyses showing a positive correlation between the frequency and diversity of AIRE-dependent TRA expression and high level expression of markers such as CD80, UEA-1 binding, and class II expression by mTECs (8, 17).

There were no obvious abnormalities in thymocyte or TEC development in newborn K5-IKK transgenic mice. Analysis of H&E stained sections revealed that the K5-IKK transgenic thymuses contained well demarcated cortical and medullary regions containing cTEC and mTEC subsets (Fig. 6A). Because thymus cellularity and thymocyte subset distribution were comparable in K5-IKK transgenic and nontransgenic newborn littermates (data not shown), the K5-IKK transgene was introduced into IKK^+/– mice, which were then intercrossed to generate IKK^–/– progeny that express the K5-IKK transgene (referred to as K5-IKK/IKK^–/– mice). Introduction of the K5-IKK transgene into newborn IKK^–/– mice resulted in a modest increase in thymus cellularity to 50% (5.1 ± 0.7 x 10⁶) of wild-type numbers. However, expression of the K5-IKK transgene in the IKK^–/– background restored medullary development as shown in H&E stained thymus sections from newborn K5-IKK/IKK^–/– mice. These medullary regions contain DCs (data not shown) and TECs (Fig. 6A). The recovery of morphologically normal K5⁺K14⁺ mTECs was not surprising given that a K5 promoter directs expression of the IKK transgene. Interestingly, the K5-IKK transgene also rescues development of the UEA-1 binding mTEC subset. Moreover, Fig. 6B shows that thymocyte depleted stromal cells from K5-IKK/IKK^–/– newborn mice express TRAs at levels comparable to those of K5-IKK cells.

View larger version (72K): [in this window] [in a new window]   FIGURE 6. The K5-IKK transgene rescues medullary development in IKK–/– mice. A, Frozen thymus sections from newborn IKK–/–, K5-IKK, and K5-IKK/IKK–/– littermates were stained with H&E (upper) at original magnification of x40 for Abs specific for K8, K5, K14, and biotinylated UEA-1 as indicated. Middle and lower panels, Original magnification, x100. Images shown are representative of thymuses from five individual mice of each genotype. B, Semiquantitative RT-PCR analysis was performed using RNA obtained from CD45 depleted stromal cells from K5-IKKa transgenic and K5-IKKa/IKKa–/– mice newborn thymuses. Three-fold serial dilutions of cDNA were used. -actin served as a loading control for each sample. A representative RT-PCR analysis from two experiments is shown.

Central tolerance is restored in IKK^–/– mice that express a K5-IKK transgene

To determine whether the K5-IKK transgene also rescued mTEC function with respect to induction of central tolerance, we transplanted K5-IKK/IKK^–/– fetal thymuses under the kidney capsule of athymic recipients. This approach was necessary because the K5-IKK/IKK^–/– mice die shortly after birth. Fig. 7A shows that well organized medullary regions containing K5⁺ and UEA-1 binding mTEC subsets were generated in the thymus grafts recovered after 6 wk. Furthermore, analysis of CD44 and CD62L expression on lymph node T cells revealed that in contrast to recipients of IKK^–/– thymus grafts, peripheral T cells generated in recipients of K5-IKK/IKK^–/– thymus grafts did not display an activated phenotype (Fig. 7B). Similar expression of Foxp3 transcripts in CD4⁺CD25⁺ splenic T cells from recipients of K5-IKK/IKK^–/– and wild-type thymus grafts suggested comparable production of peripheral Tregs (Fig. 7D). This notion was supported by immunohistochemical analysis of Foxp3-expressing cells in K5-IKK/IKK^–/– fetal thymus grafts (data not shown). The level of ANA detected in serum from recipients of K5-IKK/IKK^–/– was reduced by 60% compared with the titer of ANA in serum from recipients of IKK^–/– thymus grafts (Fig. 7C). Importantly, there was no evidence of multiorgan inflammatory infiltrates in recipients of K5-IKK/IKK^–/– thymus grafts (Fig. 7E). Taken together, these data demonstrate that expression of a K5-directed IKK transgene in IKK^–/– mice restores development of UEA-1 binding as well as K5⁺ mTECs and provides a microenvironment that supports central tolerance.

	Discussion

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This investigation demonstrates that expression of a K5 promoter-driven IKK transgene in IKK^–/– mice rescues development of the mTEC compartment and restores the ability of the thymic stromal microenvironment to generate self-tolerant T cells. Previous studies using various mutant mouse strains have shown that activation of the nonclassical NF-B signaling pathway in the thymic epithelial compartment is not only necessary for development of mTECs, particularly the UEA-1 binding subset, but also is required to induce central tolerance (19, 20, 22, 23, 31). However, the earlier studies did not determine whether the requirement for NF-B signaling pertains to one or both major mTEC subsets. The present report shows that selective expression of an IKK transgene in the K5-expressing mTEC subset rescues development of the UEA-1 binding mTEC subset and restores central tolerance in IKK^–/– mice. These data implicate the K5-positive subset in the generation of UEA-1 binding mTECs, although precise lineage relationships among TEC subsets remain obscure. In a pivotal study involving lineage tracing analysis and pharyngeal endoderm transplantation, Gordon et al. (37) demonstrated that cTECs and mTECs are derived entirely from third pharyngeal pouch endoderm confirming classical chick-quail chimera experiments (37, 38). An endoderm-derived epithelial progenitor in the fetal thymus that coexpresses MTS20 and MTS24 surface Ags was shown to generate cortical and medullary epithelial compartments (39, 40), and recent reports showing cTEC and mTEC development from single cells strengthen the argument for a common bipotent progenitor (41, 42). The data presented in this report suggest that K5-expressing mTECs are either direct precursors of UEA-1 binding cells or indirectly induce the maturation of UEA-1 binding mTECs from immature progenitors. In either case, activation of the NF-B signaling pathway is mandatory only in the K5-expressing mTEC subset.

The mTEC deficiencies that we observed in IKK^–/– mice are similar to those found in a recent report describing an independently generated IKK^–/– mouse line (31). The present investigation extends the phenotypic analysis by showing that CD11c⁺ thymic DCs are absent or severely depleted in IKK^–/– mice. Impaired TEC differentiation and autoimmune manifestations are typical features in experimental models that lack components upstream of IKK in the alternative NF-B pathway that results in RelB activation or in mice with a targeted deletion in RelB (19, 20, 22, 23). The mechanistic basis linking medullary development with the generation of self-tolerant T cells was recently clarified by studies showing that mTECs express high levels of AIRE and a diverse array of TRAs that are required to eliminate self-reactive thymocytes by negative selection (7, 8, 17, 43). Given that AIRE and TRAs are highly expressed by mTECs (8, 17), the paucity of mTECs in the IKK^–/– thymus is likely responsible for the observed reduction in AIRE and TRA expression. This interpretation is consistent with a recent report showing that NIK does not exert direct transcriptional control on genes encoding TRAs (31). Expression of CCL19 and CCL21 was also reduced in the IKK^–/– thymus. Again, this finding is not surprising because mTECs are the primary source of CCL19 and CCL21 in the thymus (33). Moreover, RelB:p52 dimers have been shown to bind the promoter regions of various chemokine loci including CCL19 (18). Thymocytes from mice that are deficient in CCL19 and CCL21 or their corresponding receptor, CCR7, accumulate in the cortex after positive selection (33). Although single positive thymocytes from these mice undergo normal maturation and are exported to the periphery, exocrinopathy develops in lacrimal and salivary glands indicating that impaired migration of single positive thymocytes into the medulla interferes with the establishment of central tolerance (44). Therefore, it is possible that a deficiency in chemokine production interferes with migration of positively selected thymocytes into the poorly developed medullary areas of IKK^–/– mice and is an additional factor that may further compromise central tolerance in this model.

IKK^–/– fetal thymus grafts placed under the kidney capsule of athymic recipients consistently generated autoimmune-like symptoms including activated peripheral T cells, high titers of ANA and prominent inflammatory infiltrates in liver and pancreas. The autoimmune phenotype developed despite the generation of DCs in the IKK^–/– thymus grafts. DCs have been shown to play an important role in establishing central tolerance to self-Ags by acquiring and cross-presenting peptides from mTECs (9). Cross-presentation is likely to be inefficient in the IKK^–/– thymus given the paucity of mTECs and corresponding reduction in TRA expression. Deficient cross-presentation could explain the failure to achieve central tolerance even though DCs develop in IKK^–/– thymus grafts. DCs also play a role in the generation of Tregs, and mTECs have recently been shown to regulate this process. In the human thymus, epithelial aggregates in the medulla (Hassall’s corpuscles) produce thymic stromal lymphopoietin that activates immature DCs to up-regulate CD80 and CD86 expression (45). Activated thymic DCs in turn promote the proliferation of CD4 single positive thymocytes, a subset of which differentiate to generate Foxp3-expressing Tregs (45). The poorly developed medullary regions in IKK^–/– thymus grafts contain few Tregs. Moreover, the diminished Foxp3 expression levels in peripheral CD4⁺ T cells in recipients of IKK^–/– thymus grafts suggest a deficiency of peripheral Tregs. Thus, a reduction in the output of Tregs as a consequence of impaired mTEC development is another factor that could contribute to the autoimmune phenotype experienced by recipients of IKK^–/– fetal thymus grafts.

Two models have been proposed to explain the expression of a wide ranging spectrum of TRAs in medullary epithelial cells. The Kyewski group (17, 46) proposes a terminal differentiation model in which TRA expression is induced as a result of derepressed gene expression during mTEC maturation. This model is based on the assumption that increasing expression of MHC class II, CD80, and UEA-1 binding molecules is coincident with mTEC maturation as well as with data showing increased expression and complexity of TRAs expressed by CD80^high or UEA-1^high mTECs (8, 17, 46). If this model is correct, then the recovery of UEA-1 binding cells in K5-IKK/IKK^–/– mice implies that IKK-mediated NF-B signaling in the K5⁺ mTEC subset initiates a maturation process culminating in the generation of mature UEA-1^high mTECs that express a diverse array of TRAs. It is not possible to ascertain from the present data whether K5⁺ mTECs are immediate precursors of UEA-1^high cells. However, a recent study showing that medullary epithelium is composed of discrete islets each of which is derived from a single precursor is consistent with the notion of a precursor-progeny relationship between the K5⁺ and UEA-1 binding mTEC subsets (47).

The Farr group (48, 49) proposes a developmental model positing that mTECs contain a population of epithelial progenitors that have not committed to a thymic epithelial lineage. As a consequence of developmental plasticity, these progenitors maintain the potential to differentiate into various epithelial lineages and therefore, express lineage-specific TRAs in response to proximal microenvironmental signals. In support of this model, Dooley et al. (50) found cystic structures in the normal thymus medulla that are surrounded by ciliated epithelial cells and express TRAs associated with respiratory epithelium. The data in the present report can be interpreted within the context of the developmental model considering our previous studies demonstrating that TEC progenitors are present in a unique subset of K8⁺K5⁺ TECs localized at the corticomedullary junction (15, 29). Assuming that multipotential precursors are also included in the K8⁺K5⁺ subset, expression of the K5-IKK transgene and consequent IKK-mediated NF-B signaling could promote the induction of individual epithelial precursors into one of several potential differentiation programs resulting in concomitant TRA expression. Presumably, this process would also induce differentiation of epithelial cells committed to a TEC fate and in the process generate UEA-1 binding cells, although not necessarily through a K5⁺K8^– mTEC intermediate.

The terminal differentiation and developmental models are not mutually exclusive. Regardless of the correct scenario, this report reveals new information on the differential requirement for IKK-mediated NF-B signaling in generating the two major medullary subsets distinguished by K5 expression and UEA-1 binding. We conclude that medulla formation and central tolerance depend on activation of the IKK-dependent alternative NF-B signaling pathway in K5⁺ epithelial cells. Activation of this signaling pathway induces K5-expressing progenitors to directly or indirectly generate the UEA-1^high mTEC subset that expresses high levels of AIRE and a diverse TRA repertoire. UEA-1^high mTECs may also play a role in thymic DC activation and generation of Tregs.

	Acknowledgments

 
We gratefully acknowledge the helpful advice and assistance provided by Carla Carter, Monica Zamisch, and Ann Griffith. We thank Kent Claypool for performing flow cytometric analysis, Joi Holcomb for assistance with figures, and Becky Brooks for manuscript preparation.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant CA16672 from the National Institutes of Health and Grant ES07784 from the National Institute on Environmental Health Sciences.

² Address correspondence and reprint requests to Dr. Ellen Richie, Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park Research Division, P.O. Box 389, 1808 Park Road 1C, Smithville, TX 78957. E-mail address: erichie{at}mdanderson.org

³ Abbreviations used in this paper: TEC, thymic epithelial cell; mTEC, medullary TEC; cTEC, cortical TEC; UEA-1, Ulex europaeus agglutinin-1; AIRE, autoimmune regulator; K5, keratin 5; IKK, IB kinase; Treg, regulatory T cell; DC, dendritic cell; TRA, tissue-restricted Ag; NIK, NF-B-inducing kinase; LTR, lymphotoxin receptor; ANA, antinuclear Ab.

Received for publication July 14, 2006. Accepted for publication October 26, 2006.

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