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International Immunology Advance Access originally published online on September 6, 2007
International Immunology 2007 19(11):1249-1260; doi:10.1093/intimm/dxm092
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Published by Oxford University Press 2007

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

Ileana Popa1, Iryna Zubkova1, Mario Medvedovic2, Tatiana Romantseva1, Howard Mostowski3, Richard Boyd4 and Marina Zaitseva1

1 Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA
2 Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA
3 Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA
4 Monash Immunology and Stem Cell Laboratories, Monash University, Melbourne, Australia

Correspondence to: M. Zaitseva; E-mail: zaitseva{at}cber.fda.gov


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Studies of HIV-1-infected individuals on anti-retroviral therapies and of patients receiving lymphoablating treatments indicate that the thymus retains restorative capacity even in adults. The contributions of the thymic epithelial cells (TECs) to the regeneration of the thymus and the identity of epithelial cell progenitors were evaluated in murine models of transient thymic atrophy followed by a complete regeneration. Using microarray approach, we analyzed the pattern of gene expression in TECs sorted from mice that were depleted of thymocytes by steroid treatment or by irradiation. The initial analysis identified significant increases in the mRNA for cMyc, Trp63 and Tcf3 transcription factors known to be expressed in early epithelial cell progenitors in tissues other than the thymus. Immunohistochemistry showed that in involuted thymuses, the cMyc and Trp63 proteins were expressed in a subset of cortical thymic epithelial cells (cTECs) that were keratin 5 positive (K5+), typifying cTEC precursors. Importantly, confocal microscopy established that epithelial cells with the phenotype of putative TEC progenitors (i.e. K5+K8+) expressed the Trp63 protein and confirmed that K5+K8+ TEC progenitors expanded significantly during atrophy and prior to the thymic regeneration. Thus, our data demonstrated for the first time that critical steps in the recovery of the adult thymus include expansion of TEC progenitors and elevated expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma. These results suggest that TEC progenitors could be reactivated in the adult thymus and, therefore, reactivation of TEC progenitors could provide a new approach for thymic reconstitution.

Keywords: epithelial cell progenitors, gene arrays, thymic stroma, thymus regeneration, transcription factors


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
The thymus plays a primary role in supplying naive T cells to the periphery by providing a microenvironment for differentiation and selection of T cells from lymphoid progenitors. Aging (1), infections with HIV-1 and with measles viruses (2), {gamma}-irradiation and in vivo administration of steroids negatively affect the function of the thymus. However, the diminished thymic activity could be restored as it was documented in AIDS patients when viremia was controlled by anti-retroviral therapies (3, 4) and in the recipients of chemotherapy upon cessation of the treatment. Thus, it is conceivable that the thymus preserves an intrinsic ability to regenerate, but the mechanisms controlling regeneration of the adult thymus are largely unknown. Studies in recipients of bone marrow transplants have shown that the age of the thymic stroma and not of the leukocyte donor was the limiting factor in the outcome of the thymic reconstitution (5). These data demonstrated the primary role of stromal cells including thymic epithelial cells (TECs) in thymic regeneration. Several cytokines and chemokines have been shown to be produced by TEC and to play a role in migration of hematopoietic precursors to the thymus (6). However, which signals are activated in TEC during adult thymus regeneration and how they contribute to the thymic function have not been investigated.

TECs together with stromal cells of non-epithelial origin create a highly organized three-dimensional support for developing T cells. Studies using mice bearing mutations in T cell genes showed that deficiencies in T cell development occur concomitantly with deficiencies in the thymic epithelium (7–12). These data strongly suggested that differentiation of TECs and maintenance of normal thymic architecture depend on the unperturbed interactions of TECs with thymocytes. Experiments using mice with mutations in the genes expressed in epithelial cells identified several transcription factors that control formation of thymic rudiment. Mice deficient in Hoxa-3 or in Pax1 transcription factors did not initiate the development of thymic primordium (13), absence of Pax9 resulted in severe defects in thymic organogenesis (14), and lack of Foxn1 blocked expansion of TECs and abrogated their capacity to attract hematopoietic precursors (15, 16). Altogether, studies using genetic manipulations in T cell or in epithelial cell genes demonstrated that development of TECs proceeds in two phases: independently from lymphoid progenitors at early stages of thymic organogenesis and under control of T cells once lymphoid progenitors colonize the fetal thymus. It has not been addressed how TEC development is affected by loss of thymocytes in genetically non-manipulated adult thymus. To answer this question, we used normal adult mice transiently depleted of thymocytes and a microarray approach to define a transcription factor cascade acting in TECs during early events in regenerating adult thymus. We demonstrated that Trp63, cMyc and Tcf3 transcription factors that are known to be involved in differentiation/proliferation of epithelial cell progenitors were up-regulated in TECs in regenerating thymic tissues and that enhanced expression of these transcription factors correlated with the expansion of putative TEC progenitors (K5+K8+).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Mice and protocol for in vivo treatments
Female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were used between 8 and 12 weeks of age. Transient thymic atrophy was induced in mice by injections with dexamethasone [(Dex) Sigma–Aldrich, St Louis, MO, USA] at 12.5 mg kg–1 intra-peritoneally in PBS or by subjecting mice to sub-lethal (2Gy) whole-body irradiation from a 137Cs source (Gammacell-40 irradiator; Atomic Energy of Canada, Ottawa, Canada) as previously described (17). Age-matching control animals received PBS. At several time points, mice were euthanized by CO2 asphyxiation, and the thymic tissues were collected. Three to seven mice were used at each time point for control and experimental groups, respectively. Mouse handling and experimental procedures were approved by Center for Biologics Evaluation and Research (CBER) animal study review committee.

Flow cytometry
For evaluation of the thymic atrophy, cell suspensions were prepared from the thymuses of individual mice and were stained with FITC-conjugated anti-CD4 (clone GK1.5) and PE-conjugated anti-CD8{alpha} mAbs (clone 53-6.7), both antibodies from BD Biosciences (San Jose, CA, USA). Twenty to fifty thousand events were collected and analyzed using FACSCaliber flow cytometer equipped with CellQuest software (BD Biosciences).

For intracellular staining, thymic tissues were treated with collagenase/DNAse I cocktail as previously described (17). Single-cell suspensions were stained using Cytofix/CytopermTM kit (BD Biosciences) and the following antibodies: rabbit anti-K5 polyclonal antibody (Covance Research, Berkeley, CA, USA) followed by PE-conjugated goat anti-rabbit IgG and Troma I mAb (anti-keratin 8, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) followed by AlexaFluor-488-conjugated donkey anti-rat IgG. Secondary reagents were purchased from Invitrogen (Carlsbad, CA, USA). Three hundred thousand events were collected and analyzed as described above.

For sorting of TECs and thymic dendritic cells (DCs), thymic tissues were pooled from three to four mice and were subjected to mechanical and then enzyme-based disruption (for details see Supplementary Methods, available at International Immunology Online).

Gene expression analysis
RNA isolation from sorted epithelial cells and amplification.
Total RNA was isolated from sorted 5000 TECs or DCs using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and subjected to two-round amplification using the MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX, USA). For DNA array experiments, aRNA was labeled with aminoallyl-UTP during the second round of amplification followed by subsequent labeling with Cy5 or with Cy3 (experimental and control samples, respectively) using CyDye Post-Labeling Reactive Dye Pack (Amersham Biosciences Corp., Piscataway, NJ, USA). Hybridization was carried out using oligo arrays containing 17 000 spots (CBER, Food and Drug Administration, Bethesda, MD, USA) for 18 h at 45°C, and following standard slide washings, the slides were scanned using GenePix 4000A scanner (Axon Instruments, Sunnyvale, CA, USA). Three hybridizations with RNA derived from the thymuses of three separate age-matched groups of animals were performed per treatment; one of three hybridizations per treatment was a dye-swap experiment.

Data normalization and statistical analysis.
Data normalization was performed in three steps for each microarray separately. First, channel-specific local background intensities were subtracted from median intensity of each channel (Cy3 and Cy5). Second, background adjusted intensities were log transformed and the differences and averages of log-transformed X1 and X2 were calculated as R = log2(X1) – log2(X2) and A = [log2(X1X2)]/2, where X1 and X2 denote the Cy5 and Cy3 intensities after subtracting local backgrounds, respectively. Third, data centering was performed by fitting the local regression model of R as a function of A (18). The difference between the observed log-ratio and the corresponding fitted value represented the normalized log-transformed gene expression ratio. Normalized log intensities for the two channels were then calculated by adding a half of the normalized ratio to A for the Cy5 channel and subtracting half of the normalized ratio from A for the Cy3 channel.

The statistical analysis was performed for each treatment by fitting the following linear model for each gene separately: Yijk = µ + Ai + Dj + Tk + {epsilon}ijk, where the Yijk corresponds to the normalized log intensity on the ith array (i = 1, ..., 9), labeled with the jth dye (j = 1 for Cy5 and j = 2 for Cy3) and for kth treatment (k = 0, 1), where k = 0 and k = 1 represented control and treated sample, respectively. µ is the overall mean log intensity, Ai is the effect of the ith array, Tk is the effect of the treatment, Dj is the effect of the jth dye and {epsilon}ijk is the random error term associate with each measurement. This model was fitted for each gene separately and P-values associated with the differential gene expression between the control sample (k = 0) and the treated sample were calculated using the empirical Bayes method after adjusting for the array and the dye effect (19). Due to a strong correlation between gene expression changes observed after treating same cell type with Dex and irradiation, the affected genes were identified by combining P-values associated with the differential gene expression between the control and experimental samples in both models using the Fisher's method (20). The combined P-values were then adjusted by calculating False Discovery Rates and genes with fdr < 0.01 (21) were considered statistically significant. Data normalization and statistical analyses were performed using the limma analysis package (http://bioinf.wehi.edu.au/limma/usersguide.pdf) and the R statistical package and programming language (22). Functional groupings for differentially expressed genes were defined using Gene Ontologies (GO) (23).

Reverse transcription–PCR analysis
RNA was purified from FACS-sorted cells and was subjected to one-round amplification as described above. Two micrograms of amplified aRNA was reverse transcribed into cDNA using oligo(dT) primers (Promega, Madison, WI, USA) and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Five microliters of cDNA was amplified by semi-quantitative PCR using Taq polymerase (Applied Biosystems, Branchburg, NJ, USA) under standard conditions. The primer pairs for PCR amplification were synthesized by Integrated DNA Technologies (Coralville, IA, USA) according to the published sequences for cMyc (24) and for GAPDH (25) or were designed using sequences reported in the GeneBank data base at the National Center for Biotechnology Information (National Institute of Health, Bethesda, MD, USA), accession numbers 009332 and 011641 for Tcf3 and Trp63, respectively. The sequences for primer pairs were as follows—cMyc: sense, 5'-GCCCAGTGAGGAATATCTGGA and anti-sense, 5'-ATCGCAGATGAAGCTCTGGT; Tcf3: sense, 5'-AGTGCAGCCATTAACCAAATC and anti-sense, 5'-TCTTCCTCTTCTTCTTCTTCCC; Trp63: sense, 5'-GCTCTTCTCCTTCTCCTTCTC and anti-sense, 5'-ACCTACTGCTTCTTTGCTAAC and GAPDH: sense, 5'-TGCACCACCAACTGCTTAG and anti-sense, 5'-GATGCAGGGATGATGTTC. PCR products (226 bp for cMyc, 241 bp for Tcf3, 176 bp for Trp63 and 176 for GAPDH) were visualized by agarose electrophoresis and quantified using the AlphaImager Imaging System (Alpha Innotech Corp., San Leandro, CA, USA). Values were normalized using signal derived from GAPDH amplification and used to calculate fold increase over control.

Immunohistology
Antibodies used for staining were as follows: rabbit polyclonal antibodies against Trp63 (H-137) that recognize all isoforms of p63 protein, goat anti-{Delta}Np63 (S-16) recognizing Trp63 isoform without transactivating domain and rabbit anti-cMyc (N-262), all three from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); rabbit anti-K5, rabbit anti-Ki67 (Novus Biologicals, Littleton, CO, USA) and mouse anti-Tcf3 mAb (Upstate Signaling Solutions, Lake Placid, NY, USA). Biotinylated secondary antibodies were purchased from Vector Laboratories (Burlingame, CA, USA).

Paraffin-embedded tissue sections were prepared from the thymuses fixed in Parafix solution (Molecular Histology Laboratory, Inc., Montgomery Village, MD, USA). Tissue sections were deparaffinized and rehydrated in 100–70% ethanol. For detection of Trp63, cMyc, Tcf3 and Ki67, rehydrated slides were first heated in 1 mM citric buffer pH 6.0 for 20 min at 95°C and cooled at room temperature for 30 min before adding of the blocking buffer (PBS, 2% normal serum, 1% BSA and 2% Tween). For detection of K5, heating step was omitted and rehydrated slides were placed in the blocking buffer. Incubations with blocking buffer, with primary antibodies, and the biotinylated secondary reagents were done for 1 h at room temperature. The reactions were developed with vectastain and Vectastain ABC-AP kits with 3,3'-diaminobezidine (DAB) and Vector Red as substrates, respectively (all reagents from Vector Laboratories). Expression of Tcf3 protein was detected using Vector M.O.M immunodetection kit (Vector Laboratories) following the manufacturer’s instructions. When double staining was performed, detection of transcription factors and of Ki67 was followed by staining for K5. The sections were counterstained with hematoxylin (Fisher Scientific, Suwanee, GA, USA). Sections were mounted with VectorShield mounting medium; the images were recorded on Zeiss Axiophot microscope with HQ camera (Photometrics, Tucson, AZ, USA) and processed using IP Lab 3.5 Software.

Confocal microscopy
Thymic tissues were fixed overnight in medium containing 0.05 M phosphate buffer, 0.1 M L-lysine pH 7.4, 2 mg ml–1 NaIO4 and 10 mg ml–1 of PFA at 4°C followed by incubation in 30% sucrose solution for 6 h as previously described (26, 27). Fixed tissues were snap frozen in OCT liquid Tissue-Tek (Sakura Finetek) and subsequently cut into 7-µm sections. For two-color microscopy, sections were stained with rabbit anti-mouse keratin 5 polyclonal antibody followed by AlexaFluor-546-conjugated goat anti-rabbit IgG and subsequently with Troma I mAb (anti-mouse keratin 8) followed by AlexaFluor-488-conjugated donkey anti-rat IgG. For three-color confocal microscopy, sections were first stained with goat anti-mouse Trp63 antibodies followed by Alexa Fluor-647 chicken anti-goat antibodies and subsequently were stained for K5 and K8. Alexa-conjugated reagents were purchased from Invitrogen. Immunofluorescence confocal microscopy was performed with a Zeiss Pascal confocal microscope. Separate images were collected for each fluorochrome and overlaid to obtain a multicolor image.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Single administration of Dex and exposure to low-dose {gamma}-irradiation induced similar kinetics of thymic atrophy in adult mice
To determine the role of TECs in the reconstitution of the adult thymus, we used two models of transient thymic atrophy previously developed in the laboratory (17). Administration of Dex and exposure to 2Gy {gamma}-irradiation induced a 2.5- to 10-fold reduction in the total number of cells in the thymuses of adult Balb/c mice 1–3 days after treatment (Fig. 1a). Thymic cellularity started to recover on day 5 and peaked (reached control levels) within 1 week (Fig. 1a and data not shown). Similarly, modest reductions in the absolute numbers of CD4–CD8– double-negative (DN) and CD4+CD8+ double-positive (DP) thymocytes were noted at day 1 and were followed by a dramatic loss of both subsets between days 2 and 3 (Fig. 1b and c). The absolute numbers of CD4+ or of CD8+ single-positive thymocytes were not affected (data not shown). DN and DP subsets started to rebound on day 5 and were completely regenerated by 1 and 2 weeks, respectively (Fig. 1b and c and data not shown). These data demonstrated that in vivo administration of 12.5 mg kg–1 of Dex and exposure to 2Gy {gamma}-irradiation induced transient thymic atrophy with similar kinetics.


Figure 1
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  		Fig. 1. Induction of transient thymic atrophy in adult mice following single administration of Dex or following exposure to small dose total body {gamma}-irradiation. (a) BALB/c mice were injected i.p. with 12.5 mg/kg of Dex (hatched bars), were exposed to 2Gy {gamma}-irradiation (dotted bars), or were injected with PBS (open bars) in control. Thymic cellularity was measured in the Dex-treated, in the irradiated, and in the age-matched PBS-treated control animals. (b, c), Absolute numbers of DN and DP thymocytes in mice following in vivo treatments. FACS analysis was performed on thymocytes isolated from mice with CD4-FITC and CD8–PE antibodies. The numbers of DN and of DP thymocytes in animals treated with PBS (open bars), with Dex (hatched bars), or in irradiated animals (dotted bars) were obtained by multiplying the percentages of CD4-CD8- thymocytes or CD4+CD8+ thymocytes by the total cellularity. Groups of three and seven mice were used at each time point in control and in treated groups, respectively. The error bars represent the mean values ± SD for individual mice. The data are representative of three separate experiments.

 
Dex treatment and {gamma}-irradiation induced similar pattern of gene expression in TECs
To search for genes that were up-regulated in TECs during thymic regeneration, we analyzed the pattern of gene expression in purified TECs collected during the peak of thymic atrophy (day 3). Thymic tissues from irradiated mice, from Dex-treated mice or from control animals were used to obtain MHC class II+ CD45– TECs by FACS sorting (Supplementary Figure S1, available at International Immunology Online). Total RNA was isolated from sorted TECs and used for hybridization with microarrays. After normalization and quality control filtering, 16 720 genes were further evaluated. Two-pass statistical analysis identified 1376 genes with fdr < 0.01 that were differentially expressed in TECs isolated from mice that received Dex or were irradiated (for complete list of 697 up-regulated and 679 down-regulated genes see Supplementary Tables S1 and S2, available at International Immunology Online). Importantly, a very strong correlation in the gene expression was observed when generated log2 ratios for differentially expressed genes in epithelial cells were compared between two models, Pearson's correlation 0.96 (Fig. 2a).


Figure 2
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  		Fig. 2. Scatter plot analysis of differentially expressed genes in TECs and in DCs purified from involuted mouse thymuses. Thymuses were isolated from mice on day 3 following in vivo injection with Dex (a, b and c) or following {gamma}-irradiation (a, b and d) and used to isolate epithelial and DCls. Total RNA was purified from pooled epithelial cells and from pooled DCs sorted from the thymuses of three to four mice per group and used for hybridization with oligoarrays. The average log2 ratios were calculated using three individual hybridizations per treatment. The data are presented as a scatter plot analyses of log2 ratios of gene expressions in the epithelial cells alone (a) and in the DCs alone (b) from Dex-treated and from irradiated mice, and of log2 ratios of gene expressions in the epithelial and DCs together from Dex-treated (c) or from irradiated (d) mice.

 
To determine whether the pattern of gene expression was unique for TECs, RNA was isolated from MHC class II+ CD45– CD11c+ DCs sorted in parallel with epithelial cells from the same enzyme-digested thymic tissues. Following hybridization with oligoarrays, log2 ratios for the genes differentially expressed in DC were compared between two treatments. Similarly to epithelial cells, a strong correlation was observed in the expression of genes in DCs between Dex-treated and irradiated animals, Pearson's correlation 0.81 (Fig. 2b; complete list of 1150 genes differentially expressed in DCs is provided in Supplementary Tables S3 and S4, available at International Immunology Online). In contrast, when epithelial cell-derived genes were compared with DC-derived genes, only 287 genes were identified that were differentially expressed with fdr < 0.01 in the thymuses of Dex-treated or of irradiated mice with no correlation in the pattern of gene expressions between TECs and DCs, Pearson's correlation –0.16 and –0.18 in the Dex-treated and irradiated mice, respectively (Fig. 2c and d). These data showed that Dex treatment and irradiation induced a very similar pattern of gene expression in TECs. However, genes that were up-regulated (down-regulated) in TECs differed from genes that were up- or down-regulated in the thymic DCs in both models.

Genes coding for transcription factors that play a role in embryogenesis, cancer and development of epithelial cell progenitors were up-regulated in TECs sorted from involuted thymuses
A strong correlation between the patterns of gene expression in TECs in both models suggested that differentiation and/or proliferation of TECs might be similarly altered by Dex and by irradiation or in response to the loss of T cells. To further elucidate how these programs were affected, a cluster of transcription factor genes that were up-regulated in TECs in Dex-treated and irradiated mice was selected using a GO based analysis (Table 1). This cluster contained a large proportion of transcription factors that are expressed during mouse embryogenesis (Hlx, Nfix and Rxrb) and were shown to regulate cardiovascular development (Sox18 and Cited2/Mrg1), development of nervous system and brain (Egr1, Egr2, Pou3f1 and GATA-2), formation of the hair follicle (Sox18, Ovol1 and Tcf3) and of the limb bud (Meis1 and Meox2), development of endothelium (Cited4), differentiation of cells in the skin (Tcf3, Ovol1 and Trp63), testes (Ovol1), morphogenesis of the eye (Meis1) and development of epithelial cells in the small intestine (Elf3 and Hlx), retina (Elf3), lung (FOXJ1) and mammary gland (Cited4). In addition, a minor group of transcription factors was identified that were expressed in many cell types in the adult tissues (CEBP{alpha}, -ß, -{delta}, Jund1 and JunB) and regulate cell cycle (CEBP and Jund1) or play a role in tumorogenesis (JunB, Rxrb and FosB). Notably, Trp63, Tcf3 and Myc genes were also up-regulated in epithelial cells isolated from involuted thymuses. Importantly, early epithelial cell progenitors in tissues other than the thymus (including skin and small intestine) were previously shown to express Trp63, Tcf3 and cMyc (28–30) suggesting a role of these transcription factors in reconstruction/maintenance of the epithelial cell network. Based on our array data and on the previously described contributions of epithelial cell precursors to thymic rebound, we hypothesized that reconstitution of the thymic epithelium might proceed through the expansion of epithelial cell precursors and is associated with up-regulation of progenitor-specific transcription factors such as Trp63, cMyc and Tcf3. Therefore, the follow-up studies were focused on the analysis of these proteins and the cell types that express them.


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  		Table 1. List of transcription factor genes up-regulated in TECs in mice following irradiation or injection with Dex

 
Trp63, cMyc and Tcf3 mRNA expressions in the sorted TECs were up-regulated during thymic atrophy and were down-regulated following regeneration
To confirm our microarray data and to determine the kinetics of Trp63, Tcf3 and cMyc mRNA expressions, semi-quantitative reverse transcription (RT)–PCR was performed using neat and diluted cDNA derived from FACS-sorted TECs at various times after treatment (Fig. 3). Miniscule levels of Trp63 and cMyc mRNA and low levels of Tcf3 mRNA were detected in TECs derived from control animals using undiluted cDNA. On day 3 following irradiation, 13- to 26-fold increases in transcription factor mRNA expressions were observed in TECs with PCR product being detected in the reactions where 27–81 times diluted cDNA was used for amplification. These high levels of gene transcription were still present at day 5. Importantly, the transcription of these genes in TECs was reduced by day 11, with more dramatic reduction observed for Trp63 and cMyc compared with Tcf3 (Fig. 3). The results of these experiments showed that Trp63, cMyc and Tcf3 gene expressions were up-regulated in TECs during peak thymic atrophy and were down-regulated at later time points when thymuses were undergoing regeneration (compare Figs 1 and 3).


Figure 3
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  		Fig. 3. Kinetics of Trp63, Tcf3 and cMyc mRNA expressions in the TECs during regeneration. Epithelial cells were FACS sorted from the thymuses of control mice and from mice on days 3, 5 and 11 after irradiation. Total RNA was extracted from sorted epithelial cells and subjected to two rounds of amplification. cDNA was prepared from amplified aRNA and used in semi-quantitative PCR with primers specific to Trp63, Tcf3, cMyc and GAPDH. Wedges indicate cDNA input (left to right: neat, diluted 1/9, 1/27 and 1/81). Table at the bottom of the figure shows the increases in the mRNA expressions for undiluted cDNA samples.

 
Expression of cMyc, Trp63 and Tcf3 transcription factor proteins was increased in involuted thymuses
Immunohistochemistry was used to determine the pattern of cMyc, Tcf3 and Trp63 protein expressions in the thymus (Fig. 4). In the normal thymic tissues, the cortical subcapsular area and the medulla contained very few Trp63-positive cells (Fig. 4a and b and data not shown). Conversely, in irradiated thymuses, Trp63 was abundantly expressed throughout the cortex (Fig. 4c and d). Few cells expressing miniscule levels of Tcf3 protein were detected in the outmost layer of the cortex in untreated thymuses (Fig. 4e and f); at the same time, strong Tcf3 staining was noted in the cortex of involuted thymuses (Fig. 4g and h). Similarly, high numbers of cMyc-positive cells were noted in the cortical areas of thymuses following irradiation and no or very few cMyc-positive cells were detected in untreated thymuses (Fig. 4i and j and data not shown). A similar pattern of transcription factor protein expression was observed in the thymuses from Dex-treated animals and confirmed our data on the increased numbers of Trp63+, cMyc+ and Tcf3+ cells in regenerating thymuses (Supplementary Figure S2 and Table S5 are available at International Immunology Online).


Figure 4
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  		Fig. 4. Trp63, Tcf3 and cMyc protein expression in the thymus during irradiation-induced atrophy. Paraffin-embedded sections of thymuses from untreated mice (a, b, e and f) and from mice at 72 h after irradiation were stained with rabbit antibodies against Trp63 (a–d) or with mouse anti-Tcf3 (e–h) or rabbit anti-cMyc (i and j) antibodies. Staining was detected by secondary reagents conjugated with HRP and the reactions were developed with DAB (shown in brown). The slides were counterstained with hematoxylin (shown in blue). Original magnifications were x10, x20, x40 and x63 and are indicated in the low right corners of the images. Images in b and f and in d, h and j show enlarged areas in inserts marked in a, e and in c, g and i, respectively.

 
Trp63 and cMyc were up-regulated within a subset of keratin 5-positive epithelial cells in the cortex of irradiated thymus
To characterize the type of epithelial cells that expressed Trp63 and cMyc, we used antibodies against keratin 5 (K5) which in the thymus is normally expressed by medullary TECs (9). As expected, in untreated thymuses, K5-expressing epithelial cells were found in the medulla and only few K5+ cells were found in the subcapsular area in the cortex (Fig. 5a). In striking contrast, K5+ cells were widely spread throughout cortex in the involuted thymuses of irradiated mice (Fig. 5b).


Figure 5
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  		Fig. 5. Trp63 and cMyc protein expression in K5-positive epithelial cells in the cortex of irradiated thymus. Paraffin-embedded sections of thymic tissues from untreated (a, c, d, g, h, k and l) and from irradiated mice (b, e, f, i, j, m and n) were stained with anti-K5 antibodies alone (a and b) or with antibodies against transcription factors Trp63 (c–f), cMyc (g–j) and against proliferation marker Ki67 (k–n) followed by anti-K5 staining (c–n). In double staining, first cMyc, Trp63 and Ki67 were detected using secondary reagents conjugated with HRP followed by development with DAB (shown in brown) (c–n); K5 expression was detected in subsequent staining using secondary reagents conjugated with alkaline phosphatase followed by Vector Red (shown in red). Nuclei were counterstained by hematoxylin (where shown). Original magnifications are indicated in the right corners. Note increased numbers of K5-positive cells that stained positively for Trp63, cMyc or Ki67 in irradiated tissues compared with control (marked with yellow arrows).

 
To determine whether K5+ TECs in the cortex of the involuted thymuses expressed Trp63, we used rabbit antibodies against K5 and goat anti-{Delta}Np63 antibodies that recognize predominant isoform of the protein (31) and that showed similar pattern of reactivity when compared with rabbit anti-p63 antibodies (data not shown). Very few if any TEC co-expressed Trp63 and K5 in the medullary and in the subcapsular regions of untreated thymuses (Fig. 5c and d). However, in the involuted thymuses, most Trp63-specific staining was detected in the nuclei of K5+ TECs located in the cortex (Fig. 5e and f). Similarly, very few cMyc+K5+ cells were detected in the subcapsular layer in the thymuses from untreated mice; as contrasted with large proportion of K5+ TECs in the cortex of irradiated thymuses expressed cMyc (Fig. 5g and h versus i and j).

To further support our notion that K5+ cortical thymic epithelial cells (cTECs) expanded during thymic atrophy, thymic tissues were co-stained with antibodies against K5 and against proliferation marker Ki67. Since Ki67 can label any type of proliferating cells, it was not surprising that Ki67+ cells were abundant in both normal and irradiated thymic tissues (Fig. 5k and m). However, the pattern of Ki67 varied: in the normal thymic tissues, very few K5+Ki67+ TECs were detected in the outmost layer of the subcapsular area (Fig. 5k and l). In comparison, numerous K5+ TECs co-expressed Ki67 in the cortex in the thymuses of irradiated mice (Fig. 5m and n). These data are in agreement with previously published observation on proliferation of epithelial cells in the cortex during thymic reconstitution in SCID model (32). Importantly, the results of our experiments extended these early observations by showing that proliferating cortical epithelial cells are K5+ and that Trp63 and cMyc transcription factors are expressed in the large proportion of K5+ cTECs (Fig. 5). The Tcf3 expression was not located in the K5+ cTECs in regenerating thymuses (data not shown). Future studies will address which type of TECs express Tcf3.

The K5+K8+ TECs were enriched in the thymuses of irradiated and of Dex-treated mice and expressed Trp63 transcription factor
In addition to medullary K5+ TECs, keratin 5 was previously shown to be expressed on a rare subset of progenitors of cortical epithelial cells, K5+K8+ cTEC precursors (9). To determine whether in our experiments, K5 was co-expressed by K8+ cTECs and whether K5+K8+ subset was increased in the thymuses of irradiated and Dex-treated mice, confocal microscopy was performed using anti-K5 and anti-K8 (Troma I) antibodies (Fig. 6). As expected, in tissues from control animals, the K8+ and K5+ TECs were restricted to the cortex and medulla, respectively, with few K5+K8+ double positive TECs positioned along the cortical–medullary junction. In contrast, in Dex-treated and in irradiated animals, the cortical areas that contained K8+ EC were somewhat reduced and, at the same time, concomitant increase in the K5+K8+ cell subset was observed within 72 h after treatment in cortical and subcapsular areas (Fig. 6). These data suggested that thymic involution as a result of Dex or irradiation treatments induced similar alterations in the stromal composition including expansion of K5+K8+ early TEC precursors.


Figure 6
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  		Fig. 6. Detection of K5+K8+ epithelial cell precursors in thymic tissues using confocal microscopy. Frozen sections were prepared from the thymuses of PBS-treated mice (a) and from mice at 24, 48 and 72 h after Dex-treatment (b–d) or irradiation (e–g). Slides were stained with rabbit polyclonal anti-K5 antibody and with Troma I rat mAb followed by Alexa Fluor-546 goat anti-rabbit (red) and Alexa Fluor-488 donkey anti-rat (green) antibodies, respectively, to visualize the medulla–cortex organization. Separate images were collected for each fluorochrome and overlaid to obtain a multicolor image. Areas where K5 staining overlaps with Troma I are shown in yellow and are marked with white arrows. Original magnification was x25.

 
To confirm these data, the thymic tissues from control, from irradiated and from Dex-treated mice were incubated with collagenase/DNAse I cocktail and the epithelial cell-enriched suspensions were subjected to intracellular staining. FACS analysis of enzyme-digested thymic tissues using anti-K5 and anti-K8 antibodies revealed a 20- to 30-fold increase in the frequency of K5+K8+ cells in the thymuses of irradiated and of Dex-treated animals (Fig. 7). In addition, tricolor confocal microscopy using antibodies against K5/K8 and against Trp63 demonstrated that K5+K8+ TECs from involuted thymuses expressed Trp63 (Fig. 8). Taken together, the results showed that TECs with a phenotype consistent with K5+K8+ cTEC precursors expanded during thymic atrophy before regeneration of the thymocyte subsets and that expanded K5+K8+ TECs expressed Trp63 transcription factor.


Figure 7
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  		Fig. 7. FACS analysis of the enzyme-digested thymic tissues. Thymuses were isolated from untreated mice and from mice 72 h after irradiation or Dex treatment. Single-cell suspensions were prepared from the thymuses using collagenase/DNAse I cocktail and were stained with rabbit polyclonal anti-K5 antibody and with Troma I rat mAb followed by PE-conjugated goat anti-rabbit and Alexa Fluor-488 donkey anti-rat reagents, respectively. Three hundred thousand events were accumulated for each sample. Numbers in upper right corners show the frequency of K5+K8+ cells. The experiment was performed three times.

 

Figure 8
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  		Fig. 8. Trp63 transcription factor is found in the K5+K8+ TEC precursors. Triple staining of frozen sections from irradiated thymuses was performed in the following order using goat anti-mouse Trp63, rat anti-mouse K8 and rabbit anti-mouse K5 antibodies and was developed with Alexa Fluor-647 chicken anti-goat, Alexa Fluor-488 goat anti-rat and Alexa Fluor-546 goat anti-rabbit antibodies, respectively. Arrows indicate K5+K8+ cells that express Trp63. Original magnification was x63, zoom 2; m, merge view.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
To search for genes that play a role in restoration of the thymic function, we employed in vivo irradiation and Dex treatment of mice to recreate a scenario of transient loss of T cells from the adult thymuses. These models were used to establish a pattern of transcription factors activated in TECs during atrophy and before restoration of thymocyte subsets. Increased expressions of genes coding for transcription factors that play a role in tumorogenesis, embryonic development and development of various epithelial tissues were detected in epithelial cells purified from the thymuses of mice during regeneration. Significantly, microarray experiments revealed increased gene transcription of Myc, Trp63 and Tcf3 transcription factors previously reported to be expressed by epithelial cell progenitors in tissues other than the thymus. RT–PCR confirmed microarray data and showed that transcription factors were up-regulated in TECs during atrophy and down-regulated after regeneration. These data suggested that T cells might exert control over the expression of transcription factors that regulate expansion/differentiation of TECs. cMyc and Trp63 proteins were detected within a subset of expanded K5+ cTECs suggesting that both transcription factors might be expressed by TEC precursors. Confocal microscopy confirmed that involuted thymuses were enriched in K5+K8+ epithelial progenitors that expressed Trp63. Thus, our data showed for the first time that regeneration of the adult thymus is preceded by expansion of TEC progenitors and that their expansion is associated with up-regulation of cMyc, Trp63 and Tcf3 in the thymic stroma.

Our previous work showed that depletion of thymocytes induced up-regulation of IL-7, Stromal Derived Factor-1 (SDF-1a), Thymus-Expressed Chemokine (TECK) and Secondary Lymphoid Tissue Chemokine (SLC) mRNA in TECs (17). These results suggested that a feedback reaction was activated in TECs by T cell depletion and was responsible for recruiting of T cell precursors to the thymus and for intra-thymic proliferation and migration of differentiating thymocytes. These data also suggested that depletion of thymocytes might activate a broad spectrum of signals in TECs that are required for restoration of the thymic stroma itself and for concomitant regeneration of the thymus. To test this hypothesis, we took advantage of high susceptibility of thymocytes to Dex and to irradiation and of relative resistance of TECs to both treatments (33, 34). The main assumption of our approach was that two treatments will generate three types of signals: genes will be induced in TECs directly in response to Dex, in response to irradiation and in response to the loss of thymocytes that occurred in both models. Statistical analysis identified nearly 700 genes that were up- or down-regulated in TECs in Dex-treated and in irradiated mice at a time of maximum atrophy (day 3) and before the onset of regeneration. High level of correlation in the obtained log2 ratios in gene expressions between two models suggested that these genes were most likely induced in TECs not by individual treatments but in response to T cell depletion. However, we cannot formally exclude that Dex or irradiation by themselves did not affect expression of any of these genes. Since epithelial cells were collected prior to regeneration, the identified up-regulated genes were used as a source to search for transcription factors that might play a role in regeneration of the epithelial cell network. The cluster of transcription factors contained genes that were previously shown to regulate differentiation of epithelial stem cells in various tissues: epidermis (cMyc) (35, 36), skin (Tcf3) (29), small intestine (cMyc) (30), hair follicle (Tcf3) (37) and also in epithelial stem cells in cervix, urogenital tract, prostate and breast (Trp63) (38). Thus, the observed increased transcriptions of Tcf3, cMyc and Trp63 genes in TECs from involuted thymuses provided strong indication that epithelial cells recovered from the thymuses prior to regeneration were enriched for epithelial cell progenitors.

Our array experiments did not detect increases in the levels of FoxN1, Hoxa-3, Pax1 and Pax9 transcription factor genes that play a major role in the formation of thymic rudiment (39). It has been suggested that early embryonic TECs, unlike adult TECs, do not require the instructive signals from hematopoietic cells (11, 39). Thus, it is possible that once the thymic rudiment is colonized with lymphoid precursors, the potential to up-regulate transcription factors that control early thymic organogenesis is irreversibly lost and cannot be reactivated in the post-natal TECs. Therefore, it is conceivable that in our experiments, depletion of T cells did not activate initial differentiation of the thymic rudiment but rather rebooted development from a stage that might be equivalent to day E12.5 when lymphoid precursors have already entered fetal thymus. This assumption was indirectly confirmed in our experiments by the observed expansion of K5+K8+ TECs that were shown to form a major TEC population in the thymic anlage at E12.5–E13.5 and in adult CD3{epsilon} transgenic mice with early blockage of T cell development (9, 11).

In normal adult mice, mature K5+ and K8+ TECs are restricted to the thymic medulla and cortex, respectively. Confocal microscopy and intracellular staining confirmed that a TEC subset that co-expressed both keratins, K5 and K8, expanded reaching 20- to 30-fold increase at 72 h after treatment. The appearance of K5 marker in cortical TECs could occur due to the up-regulation of K5 in mature K8+ TECs as a response to thymic injury, reversion of K8+ cTEC to progenitor status by expression of K5 or expansion of pre-existing K5+K8+ TECs that were previously shown to form rare subset of cortical TEC progenitors (9). Two observations supported the notion that Dex- and irradiation-induced damage of the thymus resulted in proliferation of pre-existing K5+K8+ TECs: the K5+ TECs in the cortex co-expressed Ki67 proliferation marker and the absolute numbers of epithelial cells recovered following cell sorting were higher in involuted than in control thymuses (1300 and 8700 TECs per thymus were collected after cell sorting on average from untreated and treated thymuses, respectively, data not shown). Therefore, it is more likely that enrichment of K5+K8+ TEC subset was mediated by proliferation of precursors of cTECs, although the possibility that mature K8+ TECs up-regulated K5 in response to irradiation or Dex treatment cannot be formally ruled out. It is not known what mechanism regulated proliferation of K5+K8+ cTEC precursors. It is intriguing to suggest that since DP thymocytes represent major target for Dex treatment and for irradiation, loss of DP thymocytes created space or niche in the cortex which was rapidly filled by expansion of pre-existing cTEC progenitors. Whether the K5+K8+ progenitor TECs are also capable of replenishment the medulla needs to be explored in a model of medullary depletion, e.g. relB knockout mice (40).

Two major isoforms of p63 protein that contain (TA) or lack ({Delta}N) a p53-like transactivation domain are expressed during embryogenesis with different time courses suggesting that they play distinct roles in epithelial stratification. Studies of epidermal differentiation suggested that {Delta}Np63 promotes final maturation of epidermis by inhibiting epithelial cell proliferation supported by TA p63 (41). Based on these findings it is possible to speculate that in our experiments, {Delta}Np63 detected in K5+K8+ TECs might not be involved in the expansion of TEC precursors but rather provides a switch required for the maturation of K5+K8+ TEC precursors into mature K8+K5– cTECs. In addition, in untreated thymuses, we detected very few Trp63-expressing TEC which is different from previously reported abundant expression of Trp63+ TEC in the thymuses from children aged <3 years old (42). The discrepancy between patterns of Trp63 expressions can be attributed to the age of the thymus: it is possible that in human, Trp63+ TEC precursors are present during first 3 years of life when the thymus is still under development.

The view that cortical and medullary epithelium originates from common stem cell progenitor is an enduring concept (43, 44). It has been suggested that in addition to common stem cells [such as MTS24+K5+K8+ TECs (45)], the thymus contains intermediate precursor cell types namely cortical precursor and medullary precursor. Whether regeneration of the adult thymus involves expansion of a common precursor, of its immediate progeny or of all three types is not clear. Future studies will address whether adult K5+K8+ TEC precursors that expanded during regeneration express the MTS24 marker and whether they preserve a potential to restore thymus as has been documented for K5+K8+MTS24+ from the embryo (46). It also remains to be seen whether Trp63 transcription factor is up-regulated in MTS24+ TECs (in adult or in the embryo) and whether it plays a role in the development (or activation) of K5+K8+MTS24+ TEC progenitors.

Our study showed that K5+K8+ TECs expanded in the cortex at the onset of thymic regeneration. Thus, we provide evidence that TEC progenitors previously considered to preserve a quiescent state during adulthood (47) could be reactivated and induced to proliferate in the adult thymus in order to form functional microenvironment required for complete thymic regeneration. Several strategies are now being developed for restoration of the thymus with reduced function. They include transplantation of allogeneic hematopoietic stem cells, as well as novel approaches to enhance thymopoiesis by grafting human fetal thymic tissues or by supplying hormones and cytokines that support development of T cells (reviewed in ref. 48). Demonstrating the capacity of MTS24+ TEC progenitors to generate functional thymus (45) suggested that a transfer of MTS24+-like cells could be one of the epithelial cell-focused therapies for thymic reconstitution. The results of our studies suggested that reactivation of pre-existing TEC progenitors may be instrumental for restoration of the epithelial network in the adult thymus.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary Methods, Tables S1–S5 and Figures S1–S3 are available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
This work was supported by the grant from the Office of Women's Health (FDA) 2001 and by Counter Terrorism grant 2004 from CBER, FDA to Marina Zaitseva.


   Acknowledgements
 
The authors wish to express their gratitude to Suzan Garfield and Stephen Wincovitch Sr for help with confocal microscopy and to Hana Golding and Wendy Weinberg for careful reviewing the manuscript.


   Abbreviations
 
CBER, Center for Biologics Evaluation and Research
cTEC, cortical thymic epithelial cell
DC, dendritic cell
Dex, dexamethasone
DN, double negative
DP, double positive
GO, Gene Ontology
RT, reverse transcription
TEC, thymic epithelial cell

   Notes
 
Transmitting editor: R. Flavell

Received 2 February 2007, accepted 3 August 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

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