Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus

Svetlana Ulyanchenko, Kathy E O'Neill, Tanya Medley, Alison M Farley, Harsh J Vaidya, Alistair M Cook, Natalie F Blair, C Clare Blackburn, Svetlana Ulyanchenko, Kathy E O'Neill, Tanya Medley, Alison M Farley, Harsh J Vaidya, Alistair M Cook, Natalie F Blair, C Clare Blackburn

Abstract

Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam(+)UEA1(-)Ly-51(+)PLET1(+)MHC class II(hi), which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1(+)Ly-51(+) TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Subdivision of Adult TECs by Flow Cytometry (A–C) flow cytometric analysis of TECs from 4- to 8-week-old mice for the markers shown. Plots are representative of at least three independent analyses. Plots show data after gating on (A) total EpCAM+ TECs, (B) the UEA1+ (left panel) and Ly-51+ (right panel) populations shown in (A), and (C) the PLET1+ and PLET1− subpopulations within the UEA1+ (top panels) and Ly-51+ (bottom panels) shown in (C). Gates were set on FMOs. (D) Ly-51− medullary PLET1+ co-stained with markers of mTECs as shown (scale bars, upper panels: 100 μm; lower panels: K14, K5, CLDN4, 50 μm; RAC1, 10 μm). Images show sections of thymus from 4- to 6-week-old mice after staining for the markers shown. Images are representative of at least three independent analyses. (E) Ly-51+ PLET1+ TECs present at the CMJ (scale bars, 100 μm except right hand upper panel, 15 μm). Images show sections of thymus from 4- to 6-week-old mice after staining for the markers shown. Images are representative of at least three independent analyses. See also Table S2.
Figure 2
Figure 2
Expression Profiling of Defined Adult Thymic Epithelial Cells Plots show qRT-PCR analysis of 50 sorted cells per sample for the TEC populations and genes shown. Data are normalized to the geometric mean of three housekeepers. Plet1, Aire, Cd80, Bp-1, Krt5, Ltbr, Kitl n = 4 independent experiments, all other genes, n = 3 independent cell preparations for each population. Error bars show SD. See also Table S2.
Figure 3
Figure 3
Differentiative Potential of Defined Adult Thymic Epithelial Cell Populations (A) Schematic diagram of grafting assay. Briefly, GFP+TECs were sorted and re-aggregated with mouse embryonic fibroblasts and dissociated embryonic thymic lobes. Re-aggregates were grafted under the kidney capsule for 4 weeks. (B) Sorting strategy for purification of GFP+ test populations. Plots shown are representative of more than 20 independent experiments. (C–E) Images show immunohistochemical analysis of grafts derived from the input populations shown, after staining with markers indicative of defined cTEC and mTEC populations, as shown. (C) Test GFP+ population, PLET1− mTEChi. Images show the single GFP+ area observed in two grafts analyzed. (D) Test GFP+ population, UEA1−Ly-51+PLET1− cTECs. Images show representative data from greater than three independent grafts. (E) Test GFP+ population, UEA1−Ly-51+PLET1+ TECs. Images show representative data from three independent grafts. Dotted lines in (E) show boundaries between cortex and medulla. Scale bars, 100 μm. See also Table 1, Figure S1, and Table S2.
Figure 4
Figure 4
Ly-51+PLET1+ TECs Contribute to Both mTEC and cTEC Lineages at Limiting Dilution (A) Images show immunohistochemical analysis of a single graft seeded with 90 UEA1−Ly-51+PLET1+ GFP+ input TECs, stained with the markers shown. DAPI staining (blue) indicates nuclei. Images shown are from sections taken throughout the graft, sections are numbered consecutively from top to bottom. Note that GFP+ cells contributing to all major TEC sub-lineages, including PLET1+ TECs located in the medulla and at the CMJ, were present in this graft. s, section number; M, medullary focus; C, cortical focus. Scale bars, 100 μm. Arrowheads indicate non-medullary PLET1+GFP+ TECs. (B and B′) Schematic showing distribution of GFP+ areas within graft shown in (A), along x-y axis; all sections from the graft were analyzed for the presence of GFP+ cells and the size and location of GFP foci were scored. Vertical distribution in schematic is to scale, horizontal is not. (C) Low magnification image showing relative positions of M1 and C2 in x-z plane. Scale bar, 100 μm. Maximum diameters of M1, C1, and C2 in z plane are annotated in text; maximum diameter in x and y planes are shown in (B). See also Tables S1 and S2.
Figure 5
Figure 5
The Common Progenitor Activity Is Located within the MHCIIhi Fraction of Ly-51+PLET1+ TECs (A) Graph shows contribution of test cells of the phenotypes shown to cortical and medullary TEC sublineages. x axis indicates individual grafts. y axis indicates total length of all cortical (red) and medullary (yellow) GFP+ foci in a particular graft, in micrometers. MHC class IIhiLy-51+PLET+ TECs can contribute to both cTEC and mTEC sub-lineages while MHC class IIlo/negLy-51+PLET1+ TECs can contribute only to cTECs. (B) Schematic representation showing distribution of GFP+ cells in grafts seeded with cells of the phenotypes shown. The dotted line represents the total length of the graft (1 cm represents 200 μm), for each graft, the whole graft was sectioned and each section was analyzed for the presence of GFP+ regions and the localization of the GFP+ areas to cortex and/or medulla. For the purpose of representation, information from the y and z planes are collapsed onto the x axis. Where contribution of GFP+ cells to cortical and medullary regions overlaps in this schematic, these regions were contiguous in most but not all cases. (C, C’, and D) Images show immunohistochemical analysis of grafts derived from the MHCIIhiLy-51+PLET1+ input TEC (C and C’) and MHCIIlo/negLy-51+PLET1+ (D) populations after staining with markers indicative of defined cortical and medullary TEC populations, as shown. MHCIIhiLy-51+PLET1+ input TECs, n = 4; MHCIIlo/negLy-51+PLET1+, n = 3. Images show representative data from three of four independent grafts for MHCIIhiLy-51+PLET1+ and two of three independent grafts for MHCIIlo/negLy-51+PLET1+input TECs. Arrowhead in (C′) indicates GFP+PLET1+ cell. See also Table S2.
Figure 6
Figure 6
Evidence for Stem Cell Activity among Adult Ly-51+PLET1+ TEPC Ly-51+PLET1+ TECs generate progeny present 9 months after grafting. Images show immunohistochemical analyses of a graft seeded with 1,000 UEA1−Ly-51+PLET1+GFP+ input TECs analyzed 9 months after grafting. Images show representative data from one of two independent grafts. See also Table S2.

References

    1. Bajoghli B., Aghaallaei N., Hess I., Rode I., Netuschil N., Tay B.H., Venkatesh B., Yu J.K., Kaltenbach S.L., Holland N.D. Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates. Cell. 2009;138:186–197.
    1. Benitah S.A., Frye M., Glogauer M., Watt F.M. Stem cell depletion through epidermal deletion of Rac1. Science. 2005;309:933–935.
    1. Bennett A.R., Farley A., Blair N.F., Gordon J., Sharp L., Blackburn C.C. Identification and characterization of thymic epithelial progenitor cells. Immunity. 2002;16:803–814.
    1. Billiard F., Kirshner J.R., Tait M., Danave A., Taheri S., Zhang W., Waite J.C., Olson K., Chen G., Coetzee S. Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus. Eur. J. Immunol. 2011;41:2207–2216.
    1. Bleul C.C., Corbeaux T., Reuter A., Fisch P., Mönting J.S., Boehm T. Formation of a functional thymus initiated by a postnatal epithelial progenitor cell. Nature. 2006;441:992–996.
    1. Bonfanti P., Claudinot S., Amici A.W., Farley A., Blackburn C.C., Barrandon Y. Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells. Nature. 2010;466:978–982.
    1. Bredenkamp N., Nowell C.S., Blackburn C.C. Regeneration of the aged thymus by a single transcription factor. Development. 2014;141:1627–1637.
    1. Bredenkamp N., Ulyanchenko S., O’Neill K.E., Manley N.R., Vaidya H.J., Blackburn C.C. An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts. Nat. Cell Biol. 2014;16:902–908.
    1. Buczacki S.J., Zecchini H.I., Nicholson A.M., Russell R., Vermeulen L., Kemp R., Winton D.J. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature. 2013;495:65–69.
    1. Chinn I.K., Blackburn C.C., Manley N.R., Sempowski G.D. Changes in primary lymphoid organs with aging. Semin. Immunol. 2012;24:309–320.
    1. Depreter M.G., Blair N.F., Gaskell T.L., Nowell C.S., Davern K., Pagliocca A., Stenhouse F.H., Farley A.M., Fraser A., Vrana J. Identification of Plet-1 as a specific marker of early thymic epithelial progenitor cells. Proc. Natl. Acad. Sci. USA. 2008;105:961–966.
    1. Dudakov J.A., Hanash A.M., Jenq R.R., Young L.F., Ghosh A., Singer N.V., West M.L., Smith O.M., Holland A.M., Tsai J.J. Interleukin-22 drives endogenous thymic regeneration in mice. Science. 2012;336:91–95.
    1. Frances D., Niemann C. Stem cell dynamics in sebaceous gland morphogenesis in mouse skin. Dev. Biol. 2012;363:138–146.
    1. Galy A.H., Spits H. CD40 is functionally expressed on human thymic epithelial cells. J. Immunol. 1992;149:775–782.
    1. Gill J., Malin M., Holländer G.A., Boyd R. Generation of a complete thymic microenvironment by MTS24(+) thymic epithelial cells. Nat. Immunol. 2002;3:635–642.
    1. Godfrey D.I., Izon D.J., Tucek C.L., Wilson T.J., Boyd R.L. The phenotypic heterogeneity of mouse thymic stromal cells. Immunology. 1990;70:66–74.
    1. Gordon J., Wilson V.A., Blair N.F., Sheridan J., Farley A., Wilson L., Manley N.R., Blackburn C.C. Functional evidence for a single endodermal origin for the thymic epithelium. Nat. Immunol. 2004;5:546–553.
    1. Gray D.H., Seach N., Ueno T., Milton M.K., Liston A., Lew A.M., Goodnow C.C., Boyd R.L. Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells. Blood. 2006;108:3777–3785.
    1. Gray D., Abramson J., Benoist C., Mathis D. Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire. J. Exp. Med. 2007;204:2521–2528.
    1. Hamazaki Y., Fujita H., Kobayashi T., Choi Y., Scott H.S., Matsumoto M., Minato N. Medullary thymic epithelial cells expressing Aire represent a unique lineage derived from cells expressing claudin. Nat. Immunol. 2007;8:304–311.
    1. Hozumi K., Mailhos C., Negishi N., Hirano K., Yahata T., Ando K., Zuklys S., Holländer G.A., Shima D.T., Habu S. Delta-like 4 is indispensable in thymic environment specific for T cell development. J. Exp. Med. 2008;205:2507–2513.
    1. Hu Y., Smyth G.K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009;347:70–78.
    1. Hunziker L., Benitah S.A., Braun K.M., Jensen K., McNulty K., Butler C., Potton E., Nye E., Boyd R., Laurent G. Rac1 deletion causes thymic atrophy. PLoS ONE. 2011;6:e19292.
    1. Ki S., Park D., Selden H.J., Seita J., Chung H., Kim J., Iyer V.R., Ehrlich L.I. Global transcriptional profiling reveals distinct functions of thymic stromal subsets and age-related changes during thymic involution. Cell Rep. 2014;9:402–415.
    1. Koch U., Fiorini E., Benedito R., Besseyrias V., Schuster-Gossler K., Pierres M., Manley N.R., Duarte A., Macdonald H.R., Radtke F. Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment. J. Exp. Med. 2008;205:2515–2523.
    1. Kyewski B., Klein L. A central role for central tolerance. Annu. Rev. Immunol. 2006;24:571–606.
    1. Le Douarin N.M., Jotereau F.V. Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J. Exp. Med. 1975;142:17–40.
    1. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–408.
    1. Lopes N., Sergé A., Ferrier P., Irla M. Thymic crosstalk coordinates medulla organization and T-cell tolerance induction. Front. Immunol. 2015;6:365.
    1. Manley N.R., Richie E.R., Blackburn C.C., Condie B.G., Sage J. Structure and function of the thymic microenvironment. Front. Biosci. (Landmark Ed.) 2011;16:2461–2477.
    1. Mathis L., Nicolas J.F. Cellular patterning of the vertebrate embryo. Trends Genet. 2002;18:627–635.
    1. Mayer C.E., Zuklys S., Zhanybekova S., Ohigashi I., Teh H.Y., Sansom S.N., Shikama-Dorn N., Hafen K., Macaulay I.C., Deadman M.E. Dynamic spatio-temporal contribution of single beta5t+ cortical epithelial precursors to the thymus medulla. Eur. J. Immunol. 2015 Published online December 23, 2015.
    1. Moore-Scott B.A., Opoka R., Lin S.C., Kordich J.J., Wells J.M. Identification of molecular markers that are expressed in discrete anterior-posterior domains of the endoderm from the gastrula stage to mid-gestation. Dev. Dyn. 2007;236:1997–2003.
    1. Nijhof J.G., Braun K.M., Giangreco A., van Pelt C., Kawamoto H., Boyd R.L., Willemze R., Mullenders L.H., Watt F.M., de Gruijl F.R., van Ewijk W. The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development. 2006;133:3027–3037.
    1. Nitta T., Murata S., Ueno T., Tanaka K., Takahama Y. Thymic microenvironments for T-cell repertoire formation. Adv. Immunol. 2008;99:59–94.
    1. Nowell C.S., Bredenkamp N., Tetélin S., Jin X., Tischner C., Vaidya H., Sheridan J.M., Stenhouse F.H., Heussen R., Smith A.J., Blackburn C.C. Foxn1 regulates lineage progression in cortical and medullary thymic epithelial cells but is dispensable for medullary sublineage divergence. PLoS Genet. 2011;7:e1002348.
    1. Ohigashi I., Zuklys S., Sakata M., Mayer C.E., Hamazaki Y., Minato N., Hollander G.A., Takahama Y. Adult thymic medullary epithelium is maintained and regenerated by lineage-restricted cells rather than bipotent progenitors. Cell Rep. 2015;13:1432–1443.
    1. Onder L., Nindl V., Scandella E., Chai Q., Cheng H.W., Caviezel-Firner S., Novkovic M., Bomze D., Maier R., Mair F. Alternative NF-κB signaling regulates mTEC differentiation from podoplanin-expressing presursors in the cortico-medullary junction. Eur. J. Immunol. 2015;45:2218–2231.
    1. Pellegrini G., Dellambra E., Golisano O., Martinelli E., Fantozzi I., Bondanza S., Ponzin D., McKeon F., De Luca M. p63 identifies keratinocyte stem cells. Proc. Natl. Acad. Sci. USA. 2001;98:3156–3161.
    1. Raymond K., Richter A., Kreft M., Frijns E., Janssen H., Slijper M., Praetzel-Wunder S., Langbein L., Sonnenberg A. Expression of the orphan protein Plet-1 during trichilemmal differentiation of anagen hair follicles. J. Invest. Dermatol. 2010;130:1500–1513.
    1. Reiser H., Schneeberger E.E. The costimulatory molecule B7 is expressed in the medullary region of the murine thymus. Immunology. 1994;81:532–537.
    1. Ritter M.A., Boyd R.L. Development in the thymus: it takes two to tango. Immunol. Today. 1993;14:462–469.
    1. Rode I., Boehm T. Regenerative capacity of adult cortical thymic epithelial cells. Proc. Natl. Acad. Sci. USA. 2012;109:3463–3468.
    1. Rodewald H.R., Paul S., Haller C., Bluethmann H., Blum C. Thymus medulla consisting of epithelial islets each derived from a single progenitor. Nature. 2001;414:763–768.
    1. Rossi S.W., Jenkinson W.E., Anderson G., Jenkinson E.J. Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium. Nature. 2006;441:988–991.
    1. Rossi S.W., Chidgey A.P., Parnell S.M., Jenkinson W.E., Scott H.S., Boyd R.L., Jenkinson E.J., Anderson G. Redefining epithelial progenitor potential in the developing thymus. Eur. J. Immunol. 2007;37:2411–2418.
    1. Rossi S.W., Jeker L.T., Ueno T., Kuse S., Keller M.P., Zuklys S., Gudkov A.V., Takahama Y., Krenger W., Blazar B.R., Holländer G.A. Keratinocyte growth factor (KGF) enhances postnatal T-cell development via enhancements in proliferation and function of thymic epithelial cells. Blood. 2007;109:3803–3811.
    1. Rossi S.W., Kim M.Y., Leibbrandt A., Parnell S.M., Jenkinson W.E., Glanville S.H., McConnell F.M., Scott H.S., Penninger J.M., Jenkinson E.J. RANK signals from CD4(+)3(-) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla. J. Exp. Med. 2007;204:1267–1272.
    1. Sekai M., Hamazaki Y., Minato N. Medullary thymic epithelial stem cells maintain a functional thymus to ensure lifelong central T cell tolerance. Immunity. 2014;41:753–761.
    1. Senoo M., Pinto F., Crum C.P., McKeon F. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell. 2007;129:523–536.
    1. Shakib S., Desanti G.E., Jenkinson W.E., Parnell S.M., Jenkinson E.J., Anderson G. Checkpoints in the development of thymic cortical epithelial cells. J. Immunol. 2009;182:130–137.
    1. Smith A. A glossary for stem-cell biology. Nature. 2006;441:1060.
    1. Snippert H.J., van der Flier L.G., Sato T., van Es J.H., van den Born M., Kroon-Veenboer C., Barker N., Klein A.M., van Rheenen J., Simons B.D., Clevers H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell. 2010;143:134–144.
    1. Swann J.B., Boehm T. Back to the beginning--the quest for thymic epithelial stem cells. Eur. J. Immunol. 2007;37:2364–2366.
    1. Ucar A., Ucar O., Klug P., Matt S., Brunk F., Hofmann T.G., Kyewski B. Adult thymus contains FoxN1(-) epithelial stem cells that are bipotent for medullary and cortical thymic epithelial lineages. Immunity. 2014;41:257–269.
    1. Wong K., Lister N.L., Barsanti M., Lim J.M., Hammett M.V., Khong D.M., Siatskas C., Gray D.H., Boyd R.L., Chidgey A.P. Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus. Cell Rep. 2014;8:1198–1209.

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