An Epithelial Progenitor Pool Regulates Thymus Growth William E. Jenkinson, Andrea Bacon, Andrea J. White, Graham Anderson and Eric J. Jenkinson This information is current as of October 1, 2021. J Immunol 2008; 181:6101-6108; ; doi: 10.4049/jimmunol.181.9.6101 http://www.jimmunol.org/content/181/9/6101 Downloaded from References This article cites 35 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/181/9/6101.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Epithelial Progenitor Pool Regulates Thymus Growth1

William E. Jenkinson, Andrea Bacon, Andrea J. White, Graham Anderson, and Eric J. Jenkinson2

Thymic provides an essential cellular substrate for development and selection. Gradual age-associated thymic leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers

suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a Downloaded from proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration. The Journal of Immunology, 2008, 181: 6101–6108.

ge-related changes in the contribute to damage. A key factor in this is defining the nature and persistence http://www.jimmunol.org/ an overall reduction in immune responsiveness, leading of epithelial stem or progenitor cell activity throughout the life of A to increased susceptibility to infectious disease (1). Of the thymus. During initial thymus organogenesis in the murine these changes, age-related thymus atrophy has a profound effect embryo, both cortical and medullary epithelial lineages are known on the continued generation of naive T cells and results in a pe- to arise from a common progenitor population exclusively derived ripheral T cell pool containing dominant memory T cell clones, a from the endodermal germ layer (11–13). Within the thymic pri- process that limits diversity and ultimately reduces the capacity for mordium, initial differentiation of thymic epithelium from a bipo- vaccination-based strategies (2–5). Additionally, the capacity for T tent progenitor to cortical and medullary lineages is dependent on cell-mediated immune recovery following ablative therapies and the action of the transcription factor FoxN1, and this can occur transfer declines with age in humans (6), most likely independently of interactions within the embryo (14, by guest on October 1, 2021 occurring as a result of a reduced capacity of the aged thymus to 15). Additionally, mice bearing a hypomorphic allele of FoxN1 process T cell progenitors. Several lines of evidence indicate that have demonstrated an ongoing role for this transcription factor in a reduction in the epithelial compartment of the aged thymus is a controlling thymocyte-dependent stages of thymic epithelial mat- major factor in its ability to efficiently process and select mature T uration and maintenance (16, 17). However, the ongoing persis- cells. For example, a reduction in the proportion of medullary thy- tence and identity of progenitor activity at later developmental mic epithelium essential for negative selection of autoreactive T stages into the adult remain unclear (18). Recently, two different cell clones occurs with increasing age (7), while limiting the avail- mechanisms regulating size and regenerative capacity have ability of niches directly affects thymic T cell been defined and shown to apply to different organs, even when production (8). Importantly, while the regenerative capacity of the such organs are of the same embryonic germ layer origin (19). aged thymus can be revealed in a variety of models (9, 10), neither Thus, in , reducing the epithelial progenitor pool during or- the mechanisms regulating this process nor the cell types being ganogenesis is rapidly compensated by increased expansion of the targeted are fully understood. remaining progenitors so that the organ still achieves normal size, Against this background, it is important to understand the mech- consistent with its known regenerative capacity. In contrast, re- anisms regulating the growth and size of the thymic epithelial ducing the functional progenitor pool allocated to the - compartment and its potential for regeneration following aging or forming domain of embryonic is not rescued by com- pensatory proliferation, resulting in a smaller organ, suggesting that the proliferative capacity of pancreatic epithelial progenitors is Medical Research Council Centre for Immune Regulation, Institute for Biomedical restricted and finite. Research, Medical School, University of Birmingham, Birmingham, United Kingdom In this study we have investigated whether the thymus, where Received for publication April 28, 2008. Accepted for publication August 20, 2008. the epithelium is also of an endodermal origin, conforms to either The costs of publication of this article were defrayed in part by the payment of page of these models of organ size determination. To do this, we have charges. This article must therefore be hereby marked advertisement in accordance utilized embryo fusion chimeras formed between wild-type (WT)3 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Leverhulme Early Career Fellowship (to W.E.J.) and an Medical Research Council Programme Grant (to E.J.J. and G.A.). 3 Abbreviations used in this paper: WT, wild type; E, embryonic day; EpCAM, ep- 2 Address correspondence and reprint requests to Dr. William Jenkinson, Medical ithelial cell adhesion molecule; eYFP, enhanced yellow fluorescent protein, TEC, Research Council Centre for Immune Regulation, Institute for Biomedical Research, thymic epithelial cell. Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 6102 AN EPITHELIAL PROGENITOR POOL REGULATES THYMUS GROWTH Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Generation of embryo fusion chimeric mice. A, Fusion chimeras were generated by aggregation of one 8-cell stage embryo from either donor in microwells (left panel), allowing rapid formation of a fused embryo (middle panel). Further in vitro culture resulted in formation of blastocysts of normal appearance (right panel) (magnification ϫ25). Fused blastocysts were subsequently transferred to pseudopregnant female hosts. B, Confirmation of chimera generation was performed through assessment of eYFP mosaicism in embryos of WT/FoxN1Ϫ/Ϫ mice. Additionally, chimerism was readily apparent in adult mice as indicated by overt coat chimerism in eYFPϩ(WT):eYFPϪ(WT) mice. C, Assessment of chimerism in thymic epithelium was performed through analysis of thymi from eYFPϩ(WT):eYFPϪ(WT) chimeras shown by FACS analysis. FACS plots gated on CD45Ϫ thymic stromal cells. Levels of chimerism within a single WT/FoxN1Ϫ/Ϫ adult chimera were determined within EpCAMϩ submandibular salivary (D) and within gated thymic cellular compartments of CD45ϩ (i), CD45ϪEpCAMϩ thymic epithelium (ii), and CD45ϪEpCAMϪ thymic (iii)(E). mice expressing enhanced yellow fluorescent protein (eYFP) in all genitors can compete with WT progenitors for allocation into the tissues under the control of the Rosa26 promoter and FoxN1-de- thymic-forming domain of endoderm in chimeric individuals. Due ficient nude mice (non-eYFP), allowing the contribution of each to the random variation in chimerism occurring between individual partner to various tissues in resultant individuals to be traced at mice, this provides a mechanism for determining the size of the defined stages of development. In FoxN1-deficient nude mice, nor- functional progenitor pool against its impact on the eventual size mal development of thymic epithelial progenitors is blocked at an of the thymic epithelial compartment. early stage of thymus organogenesis, although initial specification Using this approach, we show that restricting the availability of of epithelial progenitors and capacity to form a thymic rudiment a functionally competent FoxN1-expressing population within the occur via a FoxN1-independent mechanism (14). This system pro- thymic epithelial progenitor pool results in a decrease in the size of vides a model in which “sterile” developmentally incompetent pro- the thymic epithelial cellular compartment during embryonic The Journal of Immunology 6103 stages of organogenesis and that this reduction persists into the adult period. Our data support the notion that the size of the endodermal progenitor population allocated to the initial formation of the thymus has a finite capacity for expansion and can influence events in both embryonic and adult thymus, a finding that has important implications for the development of strategies aimed at regenerating the thymus following the onset of .

Materials and Methods Mice C57BL/6 nude, eYFP, CD1, and BALB/c mice were bred at the University of Birmingham, and all experiments were performed in accordance with the U.K. Home Office regulations. Adult mice were used at 4 wk of age.

Generation of embryo fusion chimeras Generation of embryo fusion chimeras was performed in line with previous reports (20). Briefly, 8-cell-stage embryos were stripped of zona pellucida using pronase. One denuded 8-cell embryo from each partner was added to a microwell and cultured in vitro at 37°C overnight in M16 media until a Downloaded from fused blastocyst was observed. Fused blastocysts were subsequently trans- ferred into 2.5-day-postcoitus pseudopregnant CD1 females. Day of blas- tocyst transfer was taken as day 3 of gestation. Generation of adult chi- meric mice was confirmed by coat color phenotype and analysis of chimerism in thymus and/or pancreatic and submandibular epithelium. http://www.jimmunol.org/ Antibodies The following Abs were used for flow cytometry: anti-CD45-PE (clone 30-F11), anti-CD4-PE (clone GK1.5), anti-CD4-allophycocyanin (clone: L3T4), anti-CD44-PE-Cy7 (clone IM7), anti-CD25-biotin (clone PC61), anti-CD3e-allophycocyanin (clone 1 45-2C11), anti-CD19-PE (clone 6D5), anti-CD8a-allophycocyanin (clone 53-6.7) (all eBioscience), strepta- vadin-PE (BD Pharmingen), and anti-EpCAM-1-Alexa Fluor 647 (epithe- lial cell adhesion molecule, clone G8.8). by guest on October 1, 2021 Cell isolation Embryonic thymi were digested using trypsin (0.25%) and EDTA (Sigma- Aldrich) to give single-cell suspensions (21). Adult thymi were cut into small pieces and incubated with collagenase dispase (2.5 mg/ml) and DNase I (1.5 ␮g/ml) (Sigma-Aldrich) at 37°C, pipetting regularly to ensure disaggregation. A subsequent brief incubation of adult thymic cells with trypsin (0.25%) at 37°C was performed to ensure full dissociation of tissue (22). Digestion of pancreas and submandibular salivary gland was per- formed using collagenase dispase (2.5 mg/ml) and DNase I (1.5 ␮g/ml).

Flow cytometry Flow cytometry was performed using a dual laser LSR I machine (BD Biosciences), with forward/side scatter gates set to exclude nonviable cells. FACS data were analyzed using FlowJo software (Tree Star). Quantitation of total adult organ cell numbers was performed using AccuCount blank particles (Spherotech).

Confocal microscopy Confocal microscopy was performed as described (23). Frozen tissue sections were stained with the following: anti-pan cytokeratin-FITC (clone C-11, Sigma-Aldrich), rabbit anti- 5 (polyclonal MK5, FIGURE 2. Embryonic WT/FoxN1Ϫ/Ϫ thymi display a defect in total Covance Research Products), anti-rabbit Ig-biotin (DakoCytomation), thymic epithelial cell numbers and size of thymocyte compartment. A, anti-CD4-Alexa Fluor 647 (clone L3T4), anti-CD8-biotin (clone CT- Thymi of E15 WT/FoxN1Ϫ/Ϫ chimeric embryos were stained for CD45 CD8b, both eBioscience), and streptavadin-Alexa Fluor 555 (Invitro- Ϫ gen). Confocal images were obtained using an LSM 510 Meta micro- and EpCAM. FACS plots gated on CD45 thymic fraction. Data are rep- scope (Zeiss) using Zeiss LSM software. resentative of three individual embryos. B, Analysis of the correlation be- tween total thymic epithelial cell numbers from both embryonic thymic lobes and the degree of nude contribution to CD45ϪEpCAMϩ thymic ep- Statistical analysis Ϫ Ϫ ithelium of E15 WT/FoxN1 / embryos (n ϭ 14). C, Analysis of the Data were evaluated using Pearson product-moment correlation coefficient correlation between total thymic epithelial cell numbers from both embry- to determine correlation between datasets. r2 values represent coefficients onic thymic lobes and the degree of nude contribution to CD45ϪEpCAMϩ of determination. A p-value of Ͻ0.05 was considered significant in all thymic epithelium of E12 WT/FoxN1Ϫ/Ϫ embryos (n ϭ 10). analyses of the significance of correlation. 6104 AN EPITHELIAL PROGENITOR POOL REGULATES THYMUS GROWTH

Results Nude-derived tissues in fusion chimeras contribute normally to most tissues but not the thymus The establishment of cortical and medullary thymic epithelial cells from bipotent progenitors represents an essential step in the gen- eration of thymic microenvironments that support the generation of a self-tolerant T cell repertoire. While it is clear that thymic epithelial cell (TEC) progenitor development involves both differ- entiation and phases of expansion, the processes acting on the pro- genitor pool to ensure normal thymus growth are not clear. To study the influence of the size of the initial thymic epithelial pro- genitor pool on thymus development, we adopted a strategy based on FoxN1-deficient embryo fusion/blastocyst complementation (24, 25). Importantly, FoxN1 regulates thymic epithelial differen- tiation in a cell-autonomous fashion (20). Moreover, while FoxN1- deficient TEC progenitors are still able to undergo initial stages of thymus organogenesis (26), in the postnatal thymus it is proposed that they may represent blocked progenitors unable to continue their differentiation program (13, 20). Thus, by generating chi- Downloaded from meric mice from a mixture of eYFP-marked WT (FoxN1-suffi- cient) and nude (FoxN1-deficient) embryos (hereafter termed WT/ FoxN1Ϫ/Ϫ mice), we assessed the impact of the presence of sterile (FoxN1-deficient) progenitors within the TEC progenitor pool on thymus development. Ϫ/Ϫ Embryo fusion chimeras were generated by fusing FoxN1 or http://www.jimmunol.org/ FoxN1ϩ/ϩ WT 8-cell embryos 1:1 with eYFP-expressing 8-cell embryos to form individual fused blastocysts (Fig. 1A), which were transferred to pseudopregnant foster mothers and allowed to develop to the required stage for analysis. Overt chimerism was evident at a gross level in both embryonic stages and in adults (Fig. 1B) and could be quantified in individual organs following disag- gregation and flow cytometry for eYFP expression in conjunction with markers of epithelial (EpCAM-1) or hematopoietic (CD45) cell populations. As described previously (25), variation in the by guest on October 1, 2021 level of chimerism was observed between individual mice in FoxN1ϩ/ϩ WT/eYFP chimeras (data not shown). However, within any one individual FoxN1ϩ/ϩ WT/eYFP mouse, chimerism was readily apparent in all tissues analyzed, including the thymus (Fig. 1C). Importantly, consistent with the inability of FoxN1Ϫ/Ϫ thy- mic epithelial cells to develop beyond the early progenitor stage, the proportion of cells of this origin in the adult thymic epithelial compartment of WT/FoxN1Ϫ/Ϫ mice was minimal even when the proportion of FoxN1Ϫ/Ϫ contribution to other tissues was high, as illustrated by comparison between chimerism seen in submandib- ular salivary gland epithelium, an organ being independent of FoxN1 function (Fig. 1D) and thymic epithelium (Fig. 1E) within a single WT/FoxN1Ϫ/Ϫ mouse. Importantly, these findings con- firm previous observations that while FoxN1Ϫ/Ϫ cells are unable to contribute normally to differentiated thymic tissue, they are able to contribute normally to other FoxN1-independent organs such as the submandibular salivary gland (25), which can therefore be used as an indicator of the level of chimerism in individual mice. Additionally, analysis of chimerism within CD45ϩ thymocytes and CD45ϪEpCAMϪ thymic mesenchyme within the same WT/ FoxN1Ϫ/Ϫ chimera (Fig. 1E) revealed no effect of loss of FoxN1 Ϫ/Ϫ function within these cellular compartments and also demonstrated FIGURE 3. Adult WT/FoxN1 thymi demonstrate a reduction in to- a similar trend of chimerism as observed within submandibular tal thymic epithelial cell numbers. The degree of FoxN1-deficient contri- bution to EpCAMϩ epithelium from submandibular salivary gland (A) and salivary gland epithelium (Fig. 1D). In support of this, comparison Ϫ ϩ pancreas (B) were correlated to total numbers of CD45 EpCAM thymic of chimerism between thymic epithelium and pancreatic epithe- epithelial cells of WT/FoxN1Ϫ/Ϫ adult mice. Within thymi of individual ϩ/ϩ lium in individual FoxN1 WT/eYFP mice revealed a similar chimeras, the degree of FoxN1-deficient contribution to CD45ϩ thymo- trend in the degree of chimerism between different organs of the cytes (C) and CD45ϪEpCAMϪ nonepithelial thymic (D) were also same mouse (data not shown). These findings are in agreement correlated to total CD45ϪEpCAMϩ thymic epithelial cell numbers. Indi- with previous studies indicating that individual embryo fusion vidual mice analyzed, n ϭ 8. The Journal of Immunology 6105 chimeras display chimerism within organs at a level comparable with the relative contributions of individual embryo partners to skin and pigment formation (25). Nude epithelial progenitors effectively compete for space in the allocation of epithelial progenitors to the thymic anlage To confirm that FoxN1Ϫ/Ϫ progenitors do initially contribute to the developing thymus in fusion chimeras and compete for “space” in the progenitor pool, we investigated the level of chimerism seen in the thymus during the early stages of thymus development. In nonchimeric FoxN1Ϫ/Ϫ mice, a thymic anlage is established from the third pharyngeal pouch endoderm. This consists of epithelial cells displaying blocked or abnormal development and persists as a discrete alymphoid structure until at least embryonic day (E)16 with no increase in size (27, 28), indicating that FoxN1Ϫ/Ϫ cells should be readily detectable in chimeric rudiments at these early stages. We therefore disaggregated WT/FoxN1Ϫ/Ϫ chimeric thy- mic lobes at E15 and looked for the presence of both FoxN1Ϫ/Ϫ (eYFPϪ) and FoxN1ϩ/ϩ WT (eYFPϩ) contributors within the ep- ithelial compartment. FoxN1Ϫ/Ϫ cells were readily detectable in Downloaded from chimeric lobes at both E12 and E15, with the range of chimerism varying between individual embryos from 10 to 50% (Fig. 2A). These findings provide direct evidence that developmentally sterile FoxN1Ϫ/Ϫ epithelial progenitors can be effectively incorporated into the developing thymic rudiment in chimeric animals. To determine whether incorporation of developmentally http://www.jimmunol.org/ blocked FoxN1Ϫ/Ϫ cells into the thymic anlage restricts the space available for WT epithelial progenitors capable of normal prolif- erative expansion, we next assessed the relationship between the total number of embryonic CD45ϪEpCAMϩ epithelial cells and the degree of FoxN1Ϫ/Ϫ contribution in individual embryonic thymi. As shown in Fig. 2B, there was a strong negative correlation between the percentage contribution of FoxN1Ϫ/Ϫ cells to the thy- mic epithelial pool and the overall number of thymic epithelial by guest on October 1, 2021 cells detected in E15 thymic lobes. A similar correlation between the total number of epithelial cells and the extent of FoxN1Ϫ/Ϫ contribution was also seen in E12 lobes (Fig. 2C), indicating that increased proliferation by WT cells cannot fully compensate for the reduction in competent progenitor number between E12 and E15. These observations confirm that the presence of FoxN1Ϫ/Ϫ cells does reduce the number of WT progenitors in the early ru- diment and provide a basis to determine whether initial reductions in the number of developmentally competent epithelial progenitors in the early thymus are compensated by the time the adult stages are reached. Functional thymic epithelial progenitor pool size determines the size of the thymic epithelial compartment Recent studies have shown that organs displaying a high level of regenerative capacity, such as liver, undergo compensatory pro- liferation in response to a reduction in the size of the initial progenitor pool (19). To investigate whether a similar compen- satory mechanism might operate within the thymus, to restore the reduction in the size of the thymic epithelial compartment observed in fetal WT/FoxN1Ϫ/Ϫ fusion chimeras, we analyzed TEC numbers in a range of adult chimeras. As FoxN1-deficient TECs are able contribute to the initial forma- FIGURE 4. A reduction in the total thymic epithelial cell pool size in tion of the embryonic thymic rudiment, but do not undergo normal WT/FoxN1Ϫ/Ϫ chimeras affects total thymocyte numbers. The total num- ϩ programs of growth and differentiation as shown above (also see Ref. bers of CD45 thymocytes were correlated to the total numbers of Ϫ ϩ Ϫ/Ϫ 14), chimerism in the adult thymus does not reflect chimerism levels CD45 EpCAM thymic epithelial cells in E15 WT/FoxN1 chimeras ϭ Ϫ/Ϫ when the thymus is first established. To overcome this problem, we (A) (individual embryos analyzed, n 19) and adult WT/FoxN1 chi- meras (B). The relationships between pancreatic EpCAMϩ epithelial compared the number of TEC in chimeric adults to the degree of ϩ FoxN1-deficient chimerism to total wet thymic mass (C) and total CD45 chimerism in a range of other tissues. As demonstrated above, this thymic cellularity (D) were compared (individual mice analyzed, n ϭ 8). provides a reflection of the degree of chimerism expected in the 6106 AN EPITHELIAL PROGENITOR POOL REGULATES THYMUS GROWTH

FIGURE 5. Adult WT/FoxN1Ϫ/Ϫ chimeric mice demonstrate normal double-negative thymocyte development and thymic epithelial organization. Ϫ Ϫ

CD4 CD8 double-negative thymocyte develop- Downloaded from mental distribution was analyzed in WT adult (A) and WT/FoxN1Ϫ/Ϫ adult thymi (B) as determined by CD44 and CD25 expression. FACS plots gated on CD4ϪCD8Ϫ cells. FACS plot is representative of six individual mice analyzed. Thymic epithelial differ- entiation and organization were analyzed by confo- ϩ cal analysis of keratin 5 medulla (red) and pan cy- http://www.jimmunol.org/ tokeratinϩ C-11 (green) epithelium in WT (C) and WT/FoxN1Ϫ/Ϫ adult thymi (D). Thymocyte organi- zation was analyzed by CD4ϩ (green) and CD8ϩ (red) staining in WT (E) and WT/FoxN1Ϫ/Ϫ adult thymi (F) (magnification ϫ25). by guest on October 1, 2021

thymus when both partners can contribute normally, reflecting the initial Collectively, these data demonstrate that, in the adult thymus, degree of chimerism in the thymus anlage. Since thymic epithe- there is a direct correlation between the degree of FoxN1-deficient lium is derived from endoderm (26), we first compared TEC num- chimerism seen across a range of tissues and the total number of bers with the level of chimerism seen in the epithelial compartment TECs. This argues that the reduction of TEC numbers observed in of other endoderm-derived organs (Fig. 3, A and B). A significant the embryonic period as a result of reducing the number of devel- correlation was observed between the total number of TECs and opmentally functional TEC progenitors allocated to the thymic an- the contribution of FoxN1-deficient cells to both submandibular lage is not overcome by compensatory proliferation by the time the salivary gland epithelium (r2 ϭϪ0.98, p ϭϽ0.00001) and pan- adult stage is reached. creatic epithelium (r2 ϭϪ0.67, p ϭ 0.01) (Fig. 3, A and B,re- spectively), with the number of TECs decreasing as the degree of Impact of thymic epithelial compartment size on thymocyte FoxN1-deficient nude chimerism increased. In support of this, a production similar correlation was also observed when TEC numbers were To investigate whether the reduced TEC frequency observed in analyzed in relation to chimerism within nonendodermal tissues WT/FoxN1Ϫ/Ϫ chimeras affects thymocyte development, we ana- including both FoxN1-deficient nude-derived thymocytes (Fig. 3C, lyzed total thymocyte numbers and compared them to total r2 ϭϪ0.88, p ϭ 0.0005) and nonepithelial thymic stromal cells epithelial cell numbers in individual thymi during embryonic (Fig. 3D, r2 ϭϪ0.92, p ϭ 0.0001). development and in the adult. At E15 (Fig. 4A) a strong correlation The Journal of Immunology 6107

Table I. Normal patterns of thymocyte development occur in the availability of stem cell niches, some of which may remain Ϫ Ϫ WT/FoxN1 / chimerasa occupied by developmentally blocked FoxN1-deficient cells, re- mains to be determined. Thymocyte Subset WT WT/FoxN1Ϫ/Ϫ Our findings also demonstrate that a reduction in the number of % CD4ϪCD8Ϫ 3.27 Ϯ 0.4 3.68 Ϯ 0.2 functional epithelial cells in intact animals has consequences for % CD4ϩCD8ϩ 81.2 Ϯ 0.5 81.37 Ϯ 5.4 the number of thymocytes produced without affecting the normal % CD4ϩCD8Ϫ 13.11 Ϯ 0.8 12.54 Ϯ 0.7 pattern of thymocyte development. This is in agreement with pre- Ϫ ϩ % CD4 CD8 2.42 Ϯ 0.2 2.41 Ϯ 0.1 vious observations where restricting the extent of epithelial pro- a Thymocyte developmental subset distribution as determined by CD4 and CD8 genitor cell proliferation by removing mesenchymal support (8) or Ϫ Ϫ expression in adult WT and WT/FoxN1 / thymi (mean Ϯ SE: WT, n ϭ 5; WT/ by knockout of the fibroblast receptor (FGFR)2iiib FoxN1Ϫ/Ϫ, n ϭ 6). on these cells (30) resulted in an overall smaller thymus with fewer thymocytes. This correlation between reduced functional epithelial cell numbers and reduced thymocyte numbers in WT/FoxN1Ϫ/Ϫ (r2 ϭ 0.64, p ϭϽ0.0001) was apparent between the total number chimeric thymi was most obvious in embryonic as compared with of TECs and total thymocyte numbers, with the latter decreasing in adult thymus where the reduction in thymocyte numbers relative to line with reductions in the number of developmentally competent epithelial cells was less marked. This difference may reflect the epithelial cells, a finding in agreement with previous observations fact that at E15, the thymocyte population is almost exclusively suggesting that thymic epithelial cell numbers limit the availability comprised of cells at the double-negative 1–3 stages, which are niches for thymocyte development (8, 29). thought to be particularly dependent on the availability of epithe- The relationship between reduced thymocyte numbers and re- lial niches or products for their development (29). Additionally, it Downloaded from duced epithelial cell numbers demonstrated a much less dramatic is possible that the requirement for thymic epithelium-derived che- correlation in the adult (Fig. 4B)(r2 ϭ 0.15). Although the corre- mokine signals required for recruitment of hematopoietic precur- lation followed the same general trend as that observed in the sors to the thymus within the embryo (31) may be more pro- embryo, it was not found to be significant ( p ϭ 0.3). Additionally, nounced in embryonic vs adult settings, leading to a more assessment of thymus mass and total thymocyte cellularity in adult pronounced defect in thymocyte numbers in embryonic WT/ Ϫ Ϫ Ϫ Ϫ WT/FoxN1 / mice compared with the degree of FoxN1 / chi- FoxN1Ϫ/Ϫ chimeras. In contrast to the reliance of DN thymocyte http://www.jimmunol.org/ merism, as determined by analysis of pancreatic epithelium, did subsets on niche availability, there is evidence that thymocyte pro- not demonstrate a significant relationship at this stage (Fig. 4, C liferation and differentiation post ␤-selection is less dependent on and D, respectively). epithelial availability (29) and this may lead to a less obvious Analysis of the developmental distribution of thymocyte subsets relationship between epithelial and thymocyte numbers as later Ϫ Ϫ in the adult thymus of WT/FoxN1 / chimeras demonstrated nor- stages of thymocyte maturation accumulate to give the dominant Ϫ Ϫ mal patterns of both CD4 CD8 double-negative I–IV subsets cortical CD4ϩCD8ϩ population seen in the adult. It remains to be compared with WT (Fig. 5, A and B) and also progression through determined whether the restriction of numbers before ␤-selection ϩ ϩ ϩ Ϫ Ϫ ϩ CD4 CD8 double-positive to CD4 CD8 and CD4 CD8 sin- has consequences for the extent of the ␣␤ TCR repertoire even by guest on October 1, 2021 gle-positive stages (Table I). Additionally, confocal analysis of though numbers may be subsequently amplified. Ϫ Ϫ WT/FoxN1 / chimeric thymi revealed normal organization and Our findings have important implications for strategies for thy- differentiation of cortical and medullary thymic epithelium as as- mic regeneration following age-related involution or for thymic sessed by cytokeratin staining (Fig. 5, C and D), as well as normal recovery after ablative therapy since they suggest that the potential distribution patterns of developing thymocytes as defined by CD4 for expansion of thymic epithelial populations in the adult may be and CD8 (Fig. 5, E and F). Taken together, these data suggest that limited. However, our findings do not exclude the possibility that despite a reduced initial thymic epithelial progenitor pool, WT/ cells with some proliferative capacity survive into the adult stages, Ϫ Ϫ FoxN1 / chimeric thymi demonstrate normal differentiation and and some studies have indicated the ability of thymic epithelial organization of thymic microenvironments and support a normal cells to respond to external stimuli and undergo expansion with program of T cell development. apparent reversal of thymic atrophy in adult mice (7, 32). In this context, although bipotent progenitors for cortical and medullary Discussion thymic epithelium have been demonstrated within embryonic and Some endodermal organs such as the liver can rapidly recover postnatal thymus (11, 13), it is as yet unclear whether such pro- from reductions in the size of the progenitor pool from which they genitors persist within the aged thymus (33). Similarly, the exis- arise to produce a normal-sized organ by the time of birth. This tence and possible persistence of lineage-restricted progenitors ability is associated with the known regenerative capacity of the ( or medulla) are still to be defined. Thus, it will be impor- adult liver and suggests that progenitor populations with a rela- tant to consider the development of alternative strategies for the tively unrestricted capacity for proliferation persist into the adult restoration of thymic function, including grafting of donor-derived stages. Conversely, the pancreas, another endoderm-derived organ, thymus tissue (34, 35) or of thymic epithelial progenitors gener- is not able to compensate for reductions in its progenitor pool and ated de novo from embryonic stem cells. lacks noticeable regenerative ability in the adult, suggesting that it arises from a progenitor pool with restricted capacity for prolifer- Disclosures ation that is mostly exhausted during organ formation (19). Our The authors have no financial conflicts of interest. present findings demonstrate that the endoderm-derived compart- ment of the thymus more closely resembles the pancreas than the References liver in its ability to compensate for reductions in the size of its 1. Gruver, A. L., L. L. Hudson, and G. D. Sempowski. 2007. of epithelial progenitor pool. These findings demonstrate that the ep- ageing. J. Pathol. 211: 144–156. ithelial progenitor pool of the thymus has a limited capacity for 2. Douek, D. C., R. D. McFarland, P. H. Keiser, E. A. Gage, J. M. Massey, B. F. Haynes, M. A. Polis, A. T. Haase, M. B. Feinberg, J. L. Sullivan, et al. 1998. proliferation within our model. Whether this limited capacity is Changes in thymic function with age and during the treatment of HIV infection. entirely intrinsic to the progenitor population or is influenced by Nature 396: 690–695. 6108 AN EPITHELIAL PROGENITOR POOL REGULATES THYMUS GROWTH

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