Prof Georg Holländer

Research Area: Developmental and Stem Cell Biology
Technology Exchange: Cell sorting, Cellular immunology, Computational biology, Ex vivo models, Flow cytometry, Immunohistochemistry, Mouse models and Transgenesis
Scientific Themes: Genes, Genetics, Epigenetics & Genomics and Molecular, Cell & Systems Biology
Keywords: Immunology, Development, Genetics, Thymus, Epithelial Cells, Autoimmunity and Immunodeficiency
Web Links:
Thymus section displaying the architectural organisation of thymic 
epithelial cells (medulla: cytokeratin 5: green; cortex: cytokeratin 8: 
blue) and mesenchymal cells (red)

Thymus section displaying the architectural organisation of thymic epithelial cells (medulla: ...

Immunohistochemical analysis (epithelial marker for the medulla: 
cytokeratin 5: green; mesenchmal marker: ERTR7) of the thymic 
microenvironment of wild type mice (left panel) and mice deficient
for the expression of Dicer in thymic epithelial cells.

Immunohistochemical analysis (epithelial marker for the medulla: cytokeratin 5: green; mesenchmal ...

Biography

Prof. Georg A Holländer was trained in both Paediatrics and Experimental Immunology in Switzerland and the U.S.  He held academic positions at Harvard Medical School, Boston, U.S. and the University of Basel, Switzerland, before he joined the University of Oxford, UK (2010). The laboratory of Developmental Immunology is part of The Weatherall Institute of Molecular Medicine (WIMM) and The Department of Paediatrics.  Due to his academic double appointment, Prof. Holländer also directs a “sister laboratory” at the Centre for Biomedicine at the University of Basel.  Both laboratories focus on the development and function of the thymus in health and disease and have over the years created different genetic mouse models to study thymus organogenesis and function and to create relevant models of human diseases related to aberrant thymus function.

Research Focus
Development and function of the thymic microenvironment: Cellular and molecular studies in health and disease

The rules and regulation of thymus development and function

The thymus is a primary lymphoid organ where T cells are generated and selected to express an antigen receptor repertoire tolerant to harmless self-antigens but reactive to potentially injurious foreign antigens. Within the boundaries of the thymic stromal environment, immigrating T cell precursors develop in an ordered maturational process that eventually gives rise to functionally and phenotypically mature T cell populations.  Separate anatomical compartments, which include the subcapsular area, the cortex, the cortical medullary junction and the medulla, comprise the thymic architecture, a structure and cellular composition well conserved throughout evolution.  Thymic epithelial cells (TECs) constitute the major component of the stroma compartment and can be subdivided according to their functional, morphological and specific antigenic features into different subpopulations including the cortical (c) and medullary (m) TEC.  The different TECs together with other stroma cells of haematopoietic (dendritic cells, macrophages, B cells) and non-haematopoietic origin (fibroblasts, endothelial cells and others) form a three dimensional meshwork.  This stromal scaffold provides the specialized microenvironment for the life-long attraction of haematopoietic precursor cells, the signals to instruct early thymocyte differentiation, the factors to guide precursor cells to different anatomical compartments, the antigenic constraints for the selection of immature T cells and the molecules necessary for the functionally mature T cells to exit to the periphery.  Though significant alterations in TEC differentiation and maintenance have been linked to debilitating states of immunodeficiency and autoimmunity, neither the molecular mechanisms responsible for regular thymus organogenesis and physiological senescence nor the pathomechanisms operational in congenital and acquired forms of thymus hypoplasia/aplasia have been completely elucidated.

The focus of the research is to delineate the molecular and cellular pathways that govern regular thymus organogenesis and function.  Specifically, the lab is interested (i) in the genetic control of the TEC differentiation and function, (ii) the phenotypic and nature and developmental potential of fetal and adult TEC precursor cells, (iii) the molecular profile and functional competence of cortical and medullary TEC; a special interest of our research is focused on understanding the epigenetic “code” that defines the regulatory principles in gene expression in TECs and the programs that determine their cellular fate.

Study

Applications are now open for
- the four year Wellcome Trust Infection, Immunology, and Translational Medicine DPhil (doctoral) programme. Further information can be found at http://www.ox.ac.uk/admissions/graduate/courses/dphil-infection-immunology-and-translational-medicine
- the three-year post-graduate fellowship offered by the Department of Paediatrics leading to a DPhil in Paediatrics. Further information can be found at http://www.paediatrics.ox.ac.uk/study/d-phil-in-paediatrics-2

Name Department Institution Country
Prof Chris Ponting MRC Functional Genomics Unit University of Oxford United Kingdom
Prof Lars Fugger Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Primo Schar Department of Biomedicine University of Basel Switzerland
Prof Bruce Blazar University of Minnesota United States
Prof Ivan Martin Tissue Engineering University of Basel Switzerland
Prof Yousuke Takahama Institute for Genome Research University of Tokushima Japan
Prof Hiroshi Kawamoto Riken Research Centre for Allergy and Immunology RIKEN Japan
Smith MJ, Reichenbach DK, Parker SL, Riddle MJ, Mitchell J, Osum KC, Mohtashami M, Stefanski HE, Fife BT, Bhandoola A et al. 2017. T cell progenitor therapy-facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction. JCI Insight, 2 (10), | Show Abstract | Read more

Infusion of in vitro-derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell-deficient (Rag1-/-) marrow with WT in vitro-generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery.

Žuklys S, Handel A, Zhanybekova S, Govani F, Keller M, Maio S, Mayer CE, Teh HY, Hafen K, Gallone G et al. 2016. Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells. Nat Immunol, 17 (10), pp. 1206-1215. | Show Abstract | Read more

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Pettitt D, Smith J, Meadows N, Arshad Z, Schuh A, DiGiusto D, Bountra C, Holländer G, Barker R, Brindley D. 2016. Regulatory barriers to the advancement of precision medicine Expert Review of Precision Medicine and Drug Development, 1 (3), pp. 319-329. | Read more

DA P, S R. 2016. The Limitations of QALY: A Literature Review Journal of Stem Cell Research & Therapy, 06 (04), | Read more

Arshad Z, Smith J, Roberts M, Lee WH, Davies B, Bure K, Hollander GA, Dopson S, Bountra C, Brindley D. 2016. Open Access Could Transform Drug Discovery: A Case Study of JQ1. Expert Opin Drug Discov, 11 (3), pp. 321-332. | Show Abstract | Read more

INTRODUCTION: The cost to develop a new drug from target discovery to market is a staggering $1.8 billion, largely due to the very high attrition rate of drug candidates and the lengthy transition times during development. Open access is an emerging model of open innovation that places no restriction on the use of information and has the potential to accelerate the development of new drugs. AREAS COVERED: To date, no quantitative assessment has yet taken place to determine the effects and viability of open access on the process of drug translation. This need is addressed within this study. The literature and intellectual property landscapes of the drug candidate JQ1, which was made available on an open access basis when discovered, and conventionally developed equivalents that were not are compared using the Web of Science and Thomson Innovation software, respectively. EXPERT OPINION: Results demonstrate that openly sharing the JQ1 molecule led to a greater uptake by a wider and more multi-disciplinary research community. A comparative analysis of the patent landscapes for each candidate also found that the broader scientific diaspora of the publically released JQ1 data enhanced innovation, evidenced by a greater number of downstream patents filed in relation to JQ1. The authors' findings counter the notion that open access drug discovery would leak commercial intellectual property. On the contrary, JQ1 serves as a test case to evidence that open access drug discovery can be an economic model that potentially improves efficiency and cost of drug discovery and its subsequent commercialization.

Mayer CE, Žuklys S, Zhanybekova S, Ohigashi I, Teh H-Y, Sansom SN, Shikama-Dorn N, Hafen K, Macaulay IC, Deadman ME et al. 2016. Dynamic spatio-temporal contribution of single β5t+ cortical epithelial precursors to the thymus medulla. Eur J Immunol, 46 (4), pp. 846-856. | Show Abstract | Read more

Intrathymic T-cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit β5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that β5t(+) TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage-tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual β5t(+) cortical progenitors located at the cortico-medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio-temporal dynamics that control the growth of the thymic medulla.

Simon AK, Hollander GA, McMichael A. 2015. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci, 282 (1821), pp. 20143085. | Show Abstract | Read more

This article reviews the development of the immune response through neonatal, infant and adult life, including pregnancy, ending with the decline in old age. A picture emerges of a child born with an immature, innate and adaptive immune system, which matures and acquires memory as he or she grows. It then goes into decline in old age. These changes are considered alongside the risks of different types of infection, autoimmune disease and malignancy.

Tang F, Gill J, Ficht X, Barthlott T, Cornils H, Schmitz-Rohmer D, Hynx D, Zhou D, Zhang L, Xue G et al. 2015. The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility Science Signaling, 8 (397), pp. ra100-ra100. | Read more

Barthlott T, Bosch AJT, Berkemeier C, Nogales-Cadenas R, Jeker LT, Keller MP, Pascual-Montano A, Holländer GA. 2015. A subpopulation of CD103(pos) ICOS(pos) Treg cells occurs at high frequency in lymphopenic mice and represents a lymph node specific differentiation stage. Eur J Immunol, 45 (6), pp. 1760-1771. | Show Abstract | Read more

Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self-reactive, chronic, and homeostatic T-cell responses. Here, we report that the increase in Treg-cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103(pos) ICOS(pos) Treg cells. Though present in the thymus, CD103(pos) ICOS(pos) Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103(pos) ICOS(pos) Treg cells is not affected by the absence of IL-7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103(pos) ICOS(pos) Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103(pos) ICOS(pos) phenotype represents a LN-specific differentiation stage arrived at by several other Treg-cell subsets. Thus, tissue-specific cues determine the overall potency of the peripheral Treg-cell pool by shaping its subset composition.

Dertschnig S, Hauri-Hohl MM, Vollmer M, Holländer GA, Krenger W. 2015. Impaired thymic expression of tissue-restricted antigens licenses the de novo generation of autoreactive CD4+ T cells in acute GVHD. Blood, 125 (17), pp. 2720-2723. | Show Abstract | Read more

During acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEC(high)) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEC(high) cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4(+) T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEC(high) cells. Our data, therefore, indicate that a functional compromise of the medullary mTEC(high) compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.

Barthlott T, Bosch AJT, Berkemeier C, Nogales-Cadenas R, Jeker LT, Keller MP, Pascual-Montano A, Holländer GA. 2015. A subpopulation of CD103<sup>pos</sup>ICOS<sup>pos</sup> Treg cells occurs at high frequency in lymphopenic mice and represents a lymph node specific differentiation stage European Journal of Immunology, 45 (6), pp. 1760-1771. | Show Abstract | Read more

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self-reactive, chronic, and homeostatic T-cell responses. Here, we report that the increase in Treg-cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103 < sup > pos < /sup > ICOS < sup > pos < /sup > Treg cells. Though present in the thymus, CD103 < sup > pos < /sup > ICOS < sup > pos < /sup > Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103 < sup > pos < /sup > ICOS < sup > pos < /sup > Treg cells is not affected by the absence of IL-7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103 < sup > pos < /sup > ICOS < sup > pos < /sup > Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103 < sup > pos < /sup > ICOS < sup > pos < /sup > phenotype represents a LN-specific differentiation stage arrived at by several other Treg-cell subsets. Thus, tissue-specific cues determine the overall potency of the peripheral Treg-cell pool by shaping its subset composition.

Salio M, Puleston DJ, Mathan TSM, Shepherd D, Stranks AJ, Adamopoulou E, Veerapen N, Besra GS, Hollander GA, Simon AK, Cerundolo V. 2014. Essential role for autophagy during invariant NKT cell development. Proc Natl Acad Sci U S A, 111 (52), pp. E5678-E5687. | Show Abstract | Read more

Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7(-/-)), thymic iNKT cell development--unlike conventional T-cell development--is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell-intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8(+) T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion.

Sansom SN, Shikama-Dorn N, Zhanybekova S, Nusspaumer G, Macaulay IC, Deadman ME, Heger A, Ponting CP, Holländer GA. 2014. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia. Genome Res, 24 (12), pp. 1918-1931. | Show Abstract | Read more

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.

Cited:

25

Scopus

Hauri-Hohl M, Zuklys S, Holländer GA, Ziegler SF. 2014. A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla Nature Immunology, 15 (6), pp. 554-561. | Show Abstract | Read more

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells. © 2014 Nature America, Inc.

Hauri-Hohl M, Zuklys S, Holländer GA, Ziegler SF. 2014. A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla. Nat Immunol, 15 (6), pp. 554-561. | Show Abstract | Read more

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

Jenkinson SR, Williams JA, Jeon H, Zhang J, Nitta T, Ohigashi I, Kruhlak M, Zuklys S, Sharrow S, Adams A et al. 2013. TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development. Proc Natl Acad Sci U S A, 110 (52), pp. 21107-21112. | Show Abstract | Read more

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra(-/-) mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.

Odaka C, Hauri-Hohl M, Takizawa K, Nishikawa Y, Yano M, Matsumoto M, Boyd R, Holländer GA. 2013. TGF-β type II receptor expression in thymic epithelial cells inhibits the development of Hassall's corpuscles in mice. Int Immunol, 25 (11), pp. 633-642. | Show Abstract | Read more

Hassall's corpuscles are concentric clusters of keratinized epithelial cells located within the thymic medulla of humans and guinea pigs but are scant in mouse and rat. They are considered to be the terminally differentiated stages of medullary thymic epithelial cells (mTECs) but the mechanisms of their origin are unclear. We have previously deleted the TGF-β type II receptor (TGFβRII) specifically in mouse TECs and reported that these mice have mitigated thymic involution and exhibit earlier reconstitution post-irradiation. In this study, we analyzed the differentiation of mTECs in the TGFβRII-knockout mice. Interestingly, the TGFβRII-knockout mice display enhanced development of Hassall's corpuscles. The expression of Aire, stromal-cell-derived factor 1 and thymic stromal lymphopoietin in the thymi of the TGFβRII-knockout mice was similar to that previously reported for the human thymus. In addition, the putative epithelial progenitor markers MTS20 and MTS24 labeled Hassall's corpuscles in normal mice, but the extent and intensity of this staining were greatly enhanced in Hassall's corpuscles of the TGFβRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall's corpuscles of the TGFβRII-knockout mice. Taken together, we suggest that TGFβRII-mediated signaling in TECs inhibits their development into Hassall's corpuscles in mice.

Shitara S, Hara T, Liang B, Wagatsuma K, Zuklys S, Holländer GA, Nakase H, Chiba T, Tani-ichi S, Ikuta K. 2013. IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRγδ+ intraepithelial lymphocytes. J Immunol, 190 (12), pp. 6173-6179. | Show Abstract | Read more

IL-7 is a cytokine essential for T cell development and survival. However, the local function of IL-7 produced by thymic epithelial cells (TECs) is poorly understood. To address this question, we generated IL-7-floxed mice and crossed them with FoxN1 promoter-driven Cre (FoxN1-Cre) mice to establish knockout mice conditionally deficient for the expression of IL-7 by TECs. We found that αβ and γδ T cells were significantly reduced in the thymus of IL-7(f/f) FoxN1-Cre mice. Proportion of mature single-positive thymocytes was increased. In lymph nodes and the spleen, the numbers of T cells were partially restored in IL-7(f/f) FoxN1-Cre mice. In addition, γδ T cells were absent from the fetal thymus and epidermis of IL-7(f/f) FoxN1-Cre mice. Furthermore, TCRγδ(+) intraepithelial lymphocytes (IELs) were significantly decreased in the small intestines of IL-7(f/f) FoxN1-Cre mice. To evaluate the function of IL-7 produced in the intestine, we crossed the IL-7(f/f) mice with villin promoter-driven Cre (Vil-Cre) mice to obtain the mice deficient in IL-7 production from intestinal epithelial cells. We observed that αβ and γδ IELs of IL-7(f/f) Vil-Cre mice were comparable to control mice. Collectively, our results suggest that TEC-derived IL-7 plays a major role in proliferation, survival, and maturation of thymocytes and is indispensable for γδ T cell development. This study also demonstrates that IL-7 produced in the thymus is essential for the development of γδ IELs and indicates the thymic origin of γδ IELs.

Ohigashi I, Zuklys S, Sakata M, Mayer CE, Zhanybekova S, Murata S, Tanaka K, Holländer GA, Takahama Y. 2013. Aire-expressing thymic medullary epithelial cells originate from β5t-expressing progenitor cells. Proc Natl Acad Sci U S A, 110 (24), pp. 9885-9890. | Show Abstract | Read more

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre-mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP-mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.

Dertschnig S, Nusspaumer G, Ivanek R, Hauri-Hohl MM, Holländer GA, Krenger W. 2013. Epithelial cytoprotection sustains ectopic expression of tissue-restricted antigens in the thymus during murine acute GVHD. Blood, 122 (5), pp. 837-841. | Show Abstract | Read more

Development of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEC(high)) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire(+)mTEC(high) to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire(+)mTEC(high) cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire(+)mTEC(high), with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.

Zuklys S, Mayer CE, Zhanybekova S, Stefanski HE, Nusspaumer G, Gill J, Barthlott T, Chappaz S, Nitta T, Dooley J et al. 2012. MicroRNAs control the maintenance of thymic epithelia and their competence for T lineage commitment and thymocyte selection. J Immunol, 189 (8), pp. 3894-3904. | Show Abstract | Read more

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

Lopez-Rios J, Speziale D, Robay D, Scotti M, Osterwalder M, Nusspaumer G, Galli A, Holländer GA, Kmita M, Zeller R. 2012. GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis. Dev Cell, 22 (4), pp. 837-848. | Show Abstract | Read more

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

Pedraza-Alva G, Mérida LB, del Rio R, Fierro NA, Cruz-Muñoz ME, Olivares N, Melchy E, Igras V, Holländer GA, Burakoff SJ, Rosenstein Y. 2011. CD43 regulates the threshold for T cell activation by targeting Cbl functions. IUBMB Life, 63 (10), pp. 940-948. | Show Abstract | Read more

T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.

Krenger W, Blazar BR, Holländer GA. 2011. Thymic T-cell development in allogeneic stem cell transplantation. Blood, 117 (25), pp. 6768-6776. | Show Abstract | Read more

Cytoreductive conditioning regimens used in the context of allogeneic hematopoietic cell transplantation (HCT) elicit deficits in innate and adaptive immunity, which predispose patients to infections. As such, transplantation outcomes depend vitally on the successful reconstruction of immune competence. Restoration of a normal peripheral T-cell pool after HCT is a slow process that requires the de novo production of naive T cells in a functionally competent thymus. However, there are several challenges to this regenerative process. Most notably, advanced age, the cytotoxic pretransplantation conditioning, and posttransplantation alloreactivity are risk factors for T-cell immune deficiency as they independently interfere with normal thymus function. Here, we discuss preclinical allogeneic HCT models and clinical observations that have contributed to a better understanding of the transplant-related thymic dysfunction. The identification of the cellular and molecular mechanisms that control regular thymopoiesis but are altered in HCT patients is expected to provide the basis for new therapies that improve the regeneration of the adaptive immune system, especially with functionally competent, naive T cells.

Lei Y, Ripen AM, Ishimaru N, Ohigashi I, Nagasawa T, Jeker LT, Bösl MR, Holländer GA, Hayashi Y, Malefyt RDW et al. 2011. Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development. J Exp Med, 208 (2), pp. 383-394. | Show Abstract | Read more

Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.

Papadopoulou AS, Dooley J, Linterman MA, Pierson W, Ucar O, Kyewski B, Zuklys S, Hollander GA, Matthys P, Gray DHD et al. 2011. The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor. Nat Immunol, 13 (2), pp. 181-187. | Show Abstract | Read more

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Holländer GA, Krenger W, Blazar BR. 2010. Emerging strategies to boost thymic function. Curr Opin Pharmacol, 10 (4), pp. 443-453. | Show Abstract | Read more

The thymus constitutes the primary lymphoid organ for the generation of T cells. Its function is particularly susceptible to various negative influences ranging from age-related involution to atrophy as a consequence of malnutrition, infection or harmful iatrogenic influences such as chemotherapy and radiation. The loss of regular thymus function significantly increases the risk for infections and cancer because of a restricted capacity for immune surveillance. In recent years, thymus-stimulatory, thymus-regenerative, and thymus-protective strategies have been developed to enhance and repair thymus function in the elderly and in individuals undergoing hematopoietic stem cell transplantation. These strategies include the use of sex steroid ablation, the administration of growth and differentiation factors, the inhibition of p53, and the transfer of T cell progenitors to alleviate the effects of thymus dysfunction and consequent T cell deficiency.

Krenger W, Hollaender GA. 2010. The role of the thymus in allogeneic haematopoietic stem cell transplantation SWISS MEDICAL WEEKLY, 140 pp. 12-22. | Read more

Berkemeier C, Hall MN, Rueegg MA, Hollaender GA. 2010. The role of the mTor pathway for the development of the mouse thymic epithelium and function SWISS MEDICAL WEEKLY, 140 (21-22), pp. 41S-41S.

Kelly RM, Goren EM, Taylor PA, Mueller SN, Stefanski HE, Osborn MJ, Scott HS, Komarova EA, Gudkov AV, Holländer GA, Blazar BR. 2010. Short-term inhibition of p53 combined with keratinocyte growth factor improves thymic epithelial cell recovery and enhances T-cell reconstitution after murine bone marrow transplantation. Blood, 115 (5), pp. 1088-1097. | Show Abstract | Read more

Myeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-beta, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. Combined before BMT KGF + PFT-beta administration additively restored numbers of cortical and medullary TECs and improved thymic function after BMT, resulting in higher numbers of donor-derived, naive peripheral CD4(+) and CD8(+) T cells. Radiation conditioning caused a loss of T-cell zone fibroblastic reticular cells (FRCs) and CCL21 expression in lymphoid stroma. KGF + PFT-beta treatment restored both FRC and CCL21 expression, findings that correlated with improved T-cell reconstitution and an enhanced immune response against Listeria monocytogenes infection. Thus, transient p53 inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery after BMT.

Vroegindeweij E, Crobach S, Itoi M, Satoh R, Zuklys S, Happe C, Germeraad WTV, Cornelissen JJ, Cupedo T, Holländer GA et al. 2010. Thymic cysts originate from Foxn1 positive thymic medullary epithelium. Mol Immunol, 47 (5), pp. 1106-1113. | Show Abstract | Read more

Thymic epithelial cells (TECs), derived from polarized two-dimensional (2D) oriented endodermal cells, are distinguished from other epithelial cells by their unique three-dimensional (3D) phenotype. However, some polarized epithelial cells remain present in the normal thymus, forming thymic cysts at the cortico-medullary junction. Here, we analyse the dynamics, origin and phenotype of such thymic cysts. In time-course experiments, we show a reverse correlation between thymic cyst expansion and the presence of thymocytes, suggesting a default pathway for the development of TECs in the absence of thymocytes. By transplanting isolated TEC populations into E15 fetal thymic lobes, we provide evidence that medullary thymic epithelial cells (mTECs), rather than cortical thymic epithelial cells (cTECs) contribute to the formation of thymic cysts. Finally, thymi of reporter mice reveal that the cysts originate from epithelia committed to a thymic fate, as indicated by the expression of Foxn1. The 2D-phenotype of cyst-lining TECs is not caused by a downregulation of Foxn1 expression, since a significant proportion of these cells in the embryonic and adult thymus continues to express Foxn1 at the protein level.

Saade M, Irla M, Yammine M, Boulanger N, Victorero G, Vincentelli R, Penninger JM, Holländer GA, Chauvet S, Nguyen C. 2010. Spatial (Tbata) expression in mature medullary thymic epithelial cells. Eur J Immunol, 40 (2), pp. 530-538. | Show Abstract | Read more

The Spatial gene is expressed in highly polarized cell types such as testis germ cells, brain neurons and thymic epithelial cells (TEC). Its expression was documented in testis and brain but poorly characterized in thymus. Here, we characterize for the first time Spatial-expressing TEC throughout ontogeny and adult mouse thymus. Spatial is expressed in thymic-fated domain by embryonic day E10.5 and persists in subcapsular, cortical, medullary epithelial cells and in MTS24(+) progenitor TEC. Using mouse strains in which thymocyte development is blocked at various stages, we show that Spatial expression is independent of thymocyte-derived signals during thymus organogenesis. Analyses on purified thymic cell subsets show that Spatial short isoforms are expressed in cortical TEC (cTEC) and mature medullary TEC (mTEC). Spatial long isoforms were detected in the same TEC population. Spatial presents a nuclear distribution specific to mature mTEC expressing UEA1 and Aire. Aire- and RANKL-deficient mice revealed that Spatial expression is drastically reduced in the thymus of these mutants. These findings reveal a critical function of Aire in regulating Spatial expression, which is compatible with promiscuous Spatial gene expression.

Krenger W, Holländer GA. 2010. The role of the thymus in allogeneic hematopoietic stem cell transplantation. Swiss Med Wkly, 140 pp. w13051. | Show Abstract | Read more

Allogeneic haematopoietic stem cell transplantation (HSCT) is used to treat an increasing number of congenital and acquired disorders of the haematopoietic system. Even though cytoreductive conditioning regimens vary in intensity, all clinically used protocols invariably cause side effects that compromise transiently or long-term the response of the natural and the adaptive immune systems. However, in the context of the reconstruction of immunity, the generation of naïve T cells constitutes a slow process, and requires a functionally competent thymus. Unfortunately, regular thymic function is frequently suppressed by transplant-related toxicities. Most notably, graft-versus-host disease (GVHD) causes a state of posttransplantation immune deficiency. Here we discuss preclinical allogeneic HSCT models and clinical observations that have contributed to a detailed understanding of the cellular and molecular mechanisms responsible for the thymic dysfunction caused by acute GVHD. An in-depth knowledge of the mechanisms that control regular thymopoiesis and, conversely, affect thymus function is expected to provide the factual basis for the design of innovative therapies to recover T-cell numbers and function following allogeneic HSCT.

Krenger W, Holländer GA. 2010. The role of the thymus in allogeneic haematopoietic stem cell transplantation Swiss Medical Weekly, 140 (JULY), | Show Abstract | Read more

Allogeneic haematopoietic stem cell transplantation (HSCT) is used to treat an increasing number of congenital and acquired disorders of the haematopoietic system. Even though cytoreductive conditioning regimens vary in intensity, all clinically used protocols invariably cause side effects that compromise transiently or long-term the response of the natural and the adaptive immune systems. However, in the context of the reconstruction of immunity, the generation of naïve T cells constitutes a slow process, and requires a functionally competent thymus. Unfortunately, regular thymic function is frequently suppressed by transplant-related toxicities. Most notably, graft-versus-host disease (GVHD) causes a state of posttransplantation immune deficiency. Here we discuss preclinical allogeneic HSCT models and clinical observations that have contributed to a detailed understanding of the cellular and molecular mechanisms responsible for the thymic dysfunction caused by acute GVHD. An in-depth knowledge of the mechanisms that control regular thymopoiesis and, conversely, affect thymus function is expected to provide the factual basis for the design of innovative therapies to recover T-cell numbers and function following allogeneic HSCT.

Fayard E, Moncayo G, Hemmings BA, Holländer GA. 2010. Phosphatidylinositol 3-kinase signaling in thymocytes: the need for stringent control. Sci Signal, 3 (135), pp. re5. | Show Abstract | Read more

The thymus serves as the primary site for the lifelong formation of new T lymphocytes; hence, it is essential for the maintenance of an effective immune system. Although thymocyte development has been widely studied, the mechanisms involved are incompletely defined. A comprehensive understanding of the molecular events that control regular thymocyte development will not only shed light on the physiological control of T cell differentiation but also probably provide insight into the pathophysiology of T cell immunodeficiencies, the molecular basis that underpins autoimmunity, and the mechanisms that instigate the formation of T cell lymphomas. Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in thymocyte development, although not all of their downstream mediators have yet been identified. Here, we discuss experimental evidence that argues for a critical role of the PI3K-phosphoinositide-dependent protein kinase (PDK1)-protein kinase B (PKB) signaling pathway in the development of both normal and malignant thymocytes, and we highlight molecules that can potentially be targeted therapeutically.

Kenins L, Gill JW, Holländer GA, Wodnar-Filipowicz A. 2010. Flt3 ligand-receptor interaction is important for maintenance of early thymic progenitor numbers in steady-state thymopoiesis. Eur J Immunol, 40 (1), pp. 81-90. | Show Abstract | Read more

T-cell production throughout life depends on efficient colonization and intrathymic expansion of BM-derived hematopoietic precursors. After irradiation-induced thymic damage, thymic recovery is facilitated by Flt3 ligand (FL), expressed by perivascular fibroblasts surrounding the thymic entry site of Flt3 receptor-positive progenitor cells. Whether intrathymic FL-Flt3 interactions play a role in steady-state replenishment of T cells remains unknown. Here, using competitive BM transplantation studies and fetal thymic organ cultures we demonstrated the continued numerical advantage of Flt3+ intrathymic T-cell precursors. Sub-kidney capsule thymic transplantation experiments, in which WT and FL-/- thymic lobes were grafted into FL-/- recipients, revealed that FL expression by the thymic microenvironment plays a role in steady-state thymopoiesis. The deficiency of the most immature thymic T-cell precursors correlated to upregulation of FL by thymic MTS15+ fibroblasts, suggesting that the number of Flt3+ progenitor cells may regulate the thymic expression of this cytokine. Together, these results show that FL expression by thymic stromal fibroblasts interacting with Flt3+ T-cell progenitors is important for the physiological maintenance of early T-cell development.

Gossens K, Naus S, Holländer GA, Ziltener HJ. 2010. Deficiency of the metalloproteinase-disintegrin ADAM8 is associated with thymic hyper-cellularity. PLoS One, 5 (9), pp. e12766. | Show Abstract | Read more

BACKGROUND: Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescence to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21. CONCLUSIONS/SIGNIFICANCE: Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration.

Gossens K, Naus S, Holländer GA, Ziltener HJ. 2010. Deficiency of the metalloproteinase-disintegrin ADAM8 is associated with thymic hyper-cellularity PLoS ONE, 5 (9), pp. 1-9. | Show Abstract | Read more

Background: Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past. Methodology/Principal Findings: Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescene to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21. Conclusions/Significance: Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration. © 2010 Gossens et al.

Shikama N, Nusspaumer G, Holländer GA. 2009. Clearing the AIRE: on the pathophysiological basis of the autoimmune polyendocrinopathy syndrome type-1. Endocrinol Metab Clin North Am, 38 (2), pp. 273-vii. | Show Abstract | Read more

Autoimmune polyendocrine syndrome type-1 clinically manifests as the triad of hypoparathyroidism, primary adrenocortical insufficiency, and chronic mucocutaneous candidiasis. Mutations in the gene that encodes the autoimmune regulator protein, AIRE, have been identified as the cause of the autoimmune polyendocrine syndrome type-1. The loss of immunologic tolerance to tissue-restricted antigens consequent to an absence of AIRE expression in the thymus results in the thymic export of autoreactive T cells that initiate autoimmunity. In this article, we discuss the role of AIRE in autoimmune polyendocrine syndrome type-1 and identify issues that still need to be addressed to fully understand the molecular pathophysiology of this complex syndrome.

Takahama Y, Saito T, Kawamoto H, Itoi M, Boyd RL, Chidgey A, Zamoyska R, Holländer GA, Anderson G, Taylor N et al. 2009. The Global Thymus Network: past, present and future. Trends Immunol, 30 (5), pp. 191-192. | Read more

Holländer GA, Peterson P. 2009. Learning to be tolerant: how T cells keep out of trouble. J Intern Med, 265 (5), pp. 541-561. | Show Abstract | Read more

A pool of immature T cells with a seemingly unrestricted repertoire of antigen specificities is generated life-long in the thymus. Amongst these cells are, however, thymocytes that express a strongly self-reactive antigen receptor and hence hold the potential to trigger autoimmunity. To prevent such an outcome, the thymus employs several independent but functionally related strategies that act in parallel to enforce self-tolerance. The deletion of strongly self-reactive thymocytes and the generation of regulatory T cells constitute the two most efficient mechanisms to induce and maintain immunological tolerance. Thymic epithelial cells of the medulla express for this purpose tissue-restricted self-antigens. This review will focus on the cellular and molecular mechanisms operative in the thymus to shape a repertoire of mature T cells tolerant to self-antigens.

Irla M, Hugues S, Gill J, Nitta T, Hikosaka Y, Williams IR, Hubert FX, Scott HS, Takahama Y, Hollaender GA, Reith W. 2009. Autoantigen-specific interactions with CD4+thymocytes control mature medullary thymic epithelial cell cellularity SWISS MEDICAL WEEKLY, 139 (9-10), pp. 16S-16S.

Zuklys S, Gill J, Keller MP, Hauri-Hohl M, Zhanybekova S, Balciunaite G, Na K-J, Jeker LT, Hafen K, Tsukamoto N et al. 2009. Stabilized beta-catenin in thymic epithelial cells blocks thymus development and function. J Immunol, 182 (5), pp. 2997-3007. | Show Abstract | Read more

Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.

Krenger W, Holländer GA. 2008. The immunopathology of thymic GVHD. Semin Immunopathol, 30 (4), pp. 439-456. | Show Abstract | Read more

The clinical success of allogeneic hematopoietic stem cell transplantation (HSCT) depends on the appropriate reconstitution of the host's immune system. While recovery of T-cell immunity may occur in transplant recipients via both thymus-dependent and thymus-independent pathways, the regeneration of a population of phenotypically naive T cells with a broad receptor repertoire relies entirely on the de novo generation of T-cells in the thymus. Preclinical models and clinical studies of allogeneic HSCT have identified the thymus as a target of graft-versus-host disease (GVHD), thus limiting T-cell regeneration. The present review focuses on recent insight into how GVHD affects thymic structure and function and how this knowledge may aid in the design of new strategies to improve T-cell reconstitution following allogeneic HSCT.

Holländer GA. 2008. Lymphoid reconstitution following hematopoietic stem cell transplantation. Of mice and men: progress made in HSCT immunobiology. Semin Immunopathol, 30 (4), pp. 369-370. | Read more

Jeker LT, Barthlott T, Keller MP, Zuklys S, Hauri-Hohl M, Deng C-X, Holländer GA. 2008. Maintenance of a normal thymic microenvironment and T-cell homeostasis require Smad4-mediated signaling in thymic epithelial cells. Blood, 112 (9), pp. 3688-3695. | Show Abstract | Read more

Signals mediated by the transforming growth factor-beta superfamily of growth factors have been implicated in thymic epithelial cell (TEC) differentiation, homeostasis, and function, but a direct reliance on these signals has not been established. Here we demonstrate that a block in canonical transforming growth factor-beta signaling by the loss of Smad4 expression in TECs leads to qualitative changes in TEC function and a progressively disorganized thymic microenvironment. Moreover, the number of thymus resident early T-lineage progenitors is severely reduced in the absence of Smad4 expression in TECs and directly correlates with extensive thymic and peripheral lymphopenia. Our observations hence place Smad4 within the signaling events in TECs that determine total thymus cellularity by controlling the number of early T-lineage progenitors.

Hozumi K, Mailhos C, Negishi N, Hirano K-I, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S. 2008. Delta-like 4 is indispensable in thymic environment specific for T cell development. J Exp Med, 205 (11), pp. 2507-2513. | Show Abstract | Read more

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Irla M, Hugues S, Gill J, Nitta T, Hikosaka Y, Williams IR, Hubert F-X, Scott HS, Takahama Y, Holländer GA, Reith W. 2008. Autoantigen-specific interactions with CD4+ thymocytes control mature medullary thymic epithelial cell cellularity. Immunity, 29 (3), pp. 451-463. | Show Abstract | Read more

Medullary thymic epithelial cells (mTECs) are specialized for inducing central immunological tolerance to self-antigens. To accomplish this, mTECs must adopt a mature phenotype characterized by expression of the autoimmune regulator Aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. The mechanisms that control mature Aire(+) mTEC development in the postnatal thymus remain poorly understood. We demonstrate here that, although either CD4(+) or CD8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mTEC population requires autoantigen-specific interactions between positively selected CD4(+) thymocytes bearing autoreactive T cell receptor (TCR) and mTECs displaying cognate self-peptide-MHC class II complexes. These interactions also involve the engagement of CD40 on mTECs by CD40L induced on the positively selected CD4(+) thymocytes. This antigen-specific TCR-MHC class II-mediated crosstalk between CD4(+) thymocytes and mTECs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mTEC population competent for ensuring central T cell tolerance.

Hauri-Hohl MM, Zuklys S, Keller MP, Jeker LT, Barthlott T, Moon AM, Roes J, Holländer GA. 2008. TGF-beta signaling in thymic epithelial cells regulates thymic involution and postirradiation reconstitution. Blood, 112 (3), pp. 626-634. | Show Abstract | Read more

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.

Kelly RM, Highfill SL, Panoskaltsis-Mortari A, Taylor PA, Boyd RL, Holländer GA, Blazar BR. 2008. Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation. Blood, 111 (12), pp. 5734-5744. | Show Abstract | Read more

Myeloablative conditioning results in thymic epithelial cell (TEC) injury, slow T-cell reconstitution, and a high risk of opportunistic infections. Keratinocyte growth factor (KGF) stimulates TEC proliferation and, when given preconditioning, reduces TEC injury. Thymocytes and TECs express androgen receptors, and exposure to androgen inhibits thymopoiesis. In this study, we have investigated whether TEC stimulation via preconditioning treatment with KGF and leuprolide acetate (Lupron), 2 clinically approved agents, given only before conditioning would circumvent the profound TEC and associated T-cell deficiency seen in allogeneic bone marrow transplant (BMT) recipients. Only combined treatment with KGF plus leuprolide acetate normalized TEC subset numbers and thymic architecture. Thymopoiesis and thymic output were supranormal, leading to the accelerated peripheral reconstitution of naive CD4 and CD8 T cells with a broad Vbeta repertoire and decreased homeostatic T-cell proliferation. Combined therapy facilitated T:B cooperativity and enabled a B-cell humoral response to a CD4 T cell-dependent neoantigen challenge soon after BMT. In vivo antigen-specific CD8 T-cell responses and clearance of a live pathogen was superior with combined versus individual agent therapy. Thus, KGF combined with androgen blockade represents a novel approach to restore thymic function and facilitates the rapid recovery of peripheral T-cell function after allogeneic BMT.

Krenger W, Holländer GA. 2008. The thymus in GVHD pathophysiology. Best Pract Res Clin Haematol, 21 (2), pp. 119-128. | Show Abstract | Read more

A favorable outcome of allogeneic hematopoietic stem-cell transplantation (HSCT) depends on the complete reconstitution of the host's immune system. While recovery of peripheral T cells occurs in transplant recipients via both thymus-dependent and thymus-independent pathways, the regeneration of a population of phenotypically naive T cells with a broad T-cell receptor (TCR) repertoire relies entirely on the de novo generation of T cells in the thymus. However, preclinical models and clinical studies of allogeneic HSCT have identified the thymus as a target of graft-versus-host disease (GVHD). The present review focuses on recent insight into how GVHD affects thymic function and how this knowledge aides the design of new strategies to improve immune reconstitution following allogeneic HSCT.

Heikenwalder M, Prinz M, Zeller N, Lang KS, Junt T, Rossi S, Tumanov A, Schmidt H, Priller J, Flatz L et al. 2008. Overexpression of lymphotoxin in T cells induces fulminant thymic involution. Am J Pathol, 172 (6), pp. 1555-1570. | Show Abstract | Read more

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Kenins L, Gill JW, Boyd RL, Holländer GA, Wodnar-Filipowicz A. 2008. Intrathymic expression of Flt3 ligand enhances thymic recovery after irradiation. J Exp Med, 205 (3), pp. 523-531. | Show Abstract | Read more

Hematopoietic stem cell transplantation (HSCT) requires conditioning treatments such as irradiation, which leads to a severely delayed recovery of T cell immunity and constitutes a major complication of this therapy. Currently, our understanding of the mechanisms regulating thymic recovery is limited. It is known that a subpopulation of bone marrow (BM)-derived thymic immigrant cells and the earliest intrathymic progenitors express the FMS-like tyrosine kinase 3 (Flt3) receptor; however, the functional significance of this expression in the thymus is not known. We used the BM transplant model to investigate the importance of Flt3 ligand (FL) for the regeneration of the T cell compartment. We show that FL is expressed in the adult mouse thymus on the surface of perivascular fibroblasts. These cells surround the proposed thymic entry site of Flt3 receptor-positive T cell progenitors. After irradiation, perivascular FL expression is up-regulated and results in an enhanced recovery of thymic cellularity. Thymic grafting experiments confirm an intrathymic requirement for FL. Collectively, these results show that thymic stromal cell-mediated FL-Flt3 receptor interactions are important in the reconstitution of thymopoiesis early after lethal irradiation and HSCT, and provide a functional relevance to the expression of the Flt3 receptor on intrathymic T cell progenitors.

Alimohammadi M, Björklund P, Hallgren A, Pöntynen N, Szinnai G, Shikama N, Keller MP, Ekwall O, Kinkel SA, Husebye ES et al. 2008. Autoimmune polyendocrine syndrome type 1 and NALP5, a parathyroid autoantigen. N Engl J Med, 358 (10), pp. 1018-1028. | Show Abstract | Read more

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disorder caused by mutations in AIRE, the autoimmune regulator gene. Though recent studies concerning AIRE deficiency have begun to elucidate the molecular pathogenesis of organ-specific autoimmunity in patients with APS-1, the autoantigen responsible for hypoparathyroidism, a hallmark of APS-1 and its most common autoimmune endocrinopathy, has not yet been identified. METHODS: We performed immunoscreening of a human parathyroid complementary DNA library, using serum samples from patients with APS-1 and hypoparathyroidism, to identify patients with reactivity to the NACHT leucine-rich-repeat protein 5 (NALP5). Subsequently, serum samples from 87 patients with APS-1 and 293 controls, including patients with other autoimmune disorders, were used to determine the frequency and specificity of autoantibodies against NALP5. In addition, the expression of NALP5 was investigated in various tissues. RESULTS: NALP5-specific autoantibodies were detected in 49% of the patients with APS-1 and hypoparathyroidism but were absent in all patients with APS-1 but without hypoparathyroidism, in all patients with other autoimmune endocrine disorders, and in all healthy controls. NALP5 was predominantly expressed in the cytoplasm of parathyroid chief cells. CONCLUSIONS: NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1.

Guimond M, Leonard WJ, Spolski R, Rossi SW, Veenstra RG, Hollander GA, Mackall CL, Blazar BR. 2008. Thymic stromal lymphopoietin is not necessary or sufficient to mediate the thymopoietic effects of keratinocyte growth factor. Blood, 111 (2), pp. 969-970. | Read more

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Hauri-Hohl MM, Zuklys S, Keller MP, Jeker LT, Barthlott T, Moon AM, Roes J, Holländer GA. 2008. TGF-<sup>2</sup>signaling in thymic epithelial cells regulates thymic involution and postirradiation reconstitution Blood, 112 (3), pp. 626-634. | Show Abstract | Read more

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in re-duced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-β signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-ßRII on thymic epithelial cells. Moreover, TGF-ß signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-ß signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation. © 2008 by The American Society of Hematology.

Jain R, Sheridan JM, Policheni A, Heinlein M, Gandolfo LC, Dewson G, Smyth GK, Sansom SN, Fu NY, Visvader JE et al. 2017. A critical epithelial survival axis regulated by MCL-1 maintains thymic function in mice. Blood, 130 (23), pp. 2504-2515. | Show Abstract | Read more

T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.

Pettitt D, Arshad Z, Davies B, Smith J, French A, Cole D, Bure K, Dopson S, DiGiusto D, Karp J et al. 2017. An assessment of the factors affecting the commercialization of cell-based therapeutics: a systematic review protocol. Syst Rev, 6 (1), pp. 120. | Show Abstract | Read more

BACKGROUND: Cellular-based therapies represent a platform technology within the rapidly expanding field of regenerative medicine and are distinct from conventional therapeutics-offering a unique approach to managing what were once considered untreatable diseases. Despite a significant increase in basic science activity within the cell therapy arena, alongside a growing portfolio of cell therapy trials and promising investment, the translation of cellular-based therapeutics from "bench to bedside" remains challenging, and the number of industry products available for widespread clinical use remains comparatively low. This systematic review identifies unique intrinsic and extrinsic barriers in the cell-based therapy domain. METHODS/DESIGN: Eight electronic databases will be searched, specifically Medline, EMBASE (OvidSP), BIOSIS & Web of Science, Cochrane Library & HEED, EconLit (ProQuest), WHOLIS WHO Library Database, PAIS International (ProQuest), and Scopus. Addition to this gray literature was searched by manually reviewing relevant work. All identified articles will be subjected for review by two authors who will decide whether or not each article passes our inclusion/exclusion criteria. Eligible papers will subsequently be reviewed, and key data extracted into a pre-designed data extraction scorecard. An assessment of the perceived impact of broad commercial barriers to the adoption of cell-based therapies will be conducted. These broad categories will include manufacturing, regulation and intellectual property, reimbursement, clinical trials, clinical adoption, ethics, and business models. This will inform further discussion in the review. There is no PROSPERO registration number. DISCUSSION: Through a systematic search and appraisal of available literature, this review will identify key challenges in the commercialization pathway of cellular-based therapeutics and highlights significant barriers impeding successful clinical adoption. This will aid in creating an adaptable, acceptable, and harmonized approach supported by apposite regulatory frameworks and pertinent expertise throughout the respective stages of the adoption cycle to facilitate the adoption of new products and technologies in the industry.

Satoh R, Kakugawa K, Yasuda T, Yoshida H, Sibilia M, Katsura Y, Levi B, Abramson J, Koseki Y, Koseki H et al. 2016. Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells. PLoS Genet, 12 (1), pp. e1005776. | Show Abstract | Read more

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

Ohigashi I, Zuklys S, Sakata M, Mayer CE, Hamazaki Y, Minato N, Hollander GA, Takahama Y. 2015. Adult Thymic Medullary Epithelium Is Maintained and Regenerated by Lineage-Restricted Cells Rather Than Bipotent Progenitors. Cell Rep, 13 (7), pp. 1432-1443. | Show Abstract | Read more

Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in T cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have been reported. Here, we report in vivo fate-mapping analysis of cells that transcribe β5t, a cTEC trait expressed in bipotent progenitors, during a given period in mice. We show that, in adult mice, most mTECs are derived from progenitors that transcribe β5t during embryogenesis and the neonatal period up to 1 week of age. The contribution of adult β5t(+) progenitors was minor even during injury-triggered regeneration. Our results further demonstrate that adult mTEC-restricted progenitors are derived from perinatal β5t(+) progenitors. These results indicate that the adult thymic medullary epithelium is maintained and regenerated by mTEC-lineage cells that pass beyond the bipotent stage during early ontogeny.

Shitara S, Hara T, Liang B, Wagatsuma K, Zuklys S, Holländer GA, Nakase H, Chiba T, Tani-ichi S, Ikuta K. 2013. IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRγδ+ intraepithelial lymphocytes. J Immunol, 190 (12), pp. 6173-6179. | Show Abstract | Read more

IL-7 is a cytokine essential for T cell development and survival. However, the local function of IL-7 produced by thymic epithelial cells (TECs) is poorly understood. To address this question, we generated IL-7-floxed mice and crossed them with FoxN1 promoter-driven Cre (FoxN1-Cre) mice to establish knockout mice conditionally deficient for the expression of IL-7 by TECs. We found that αβ and γδ T cells were significantly reduced in the thymus of IL-7(f/f) FoxN1-Cre mice. Proportion of mature single-positive thymocytes was increased. In lymph nodes and the spleen, the numbers of T cells were partially restored in IL-7(f/f) FoxN1-Cre mice. In addition, γδ T cells were absent from the fetal thymus and epidermis of IL-7(f/f) FoxN1-Cre mice. Furthermore, TCRγδ(+) intraepithelial lymphocytes (IELs) were significantly decreased in the small intestines of IL-7(f/f) FoxN1-Cre mice. To evaluate the function of IL-7 produced in the intestine, we crossed the IL-7(f/f) mice with villin promoter-driven Cre (Vil-Cre) mice to obtain the mice deficient in IL-7 production from intestinal epithelial cells. We observed that αβ and γδ IELs of IL-7(f/f) Vil-Cre mice were comparable to control mice. Collectively, our results suggest that TEC-derived IL-7 plays a major role in proliferation, survival, and maturation of thymocytes and is indispensable for γδ T cell development. This study also demonstrates that IL-7 produced in the thymus is essential for the development of γδ IELs and indicates the thymic origin of γδ IELs.

Ohigashi I, Zuklys S, Sakata M, Mayer CE, Zhanybekova S, Murata S, Tanaka K, Holländer GA, Takahama Y. 2013. Aire-expressing thymic medullary epithelial cells originate from β5t-expressing progenitor cells. Proc Natl Acad Sci U S A, 110 (24), pp. 9885-9890. | Show Abstract | Read more

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre-mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP-mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.

Dertschnig S, Nusspaumer G, Ivanek R, Hauri-Hohl MM, Holländer GA, Krenger W. 2013. Epithelial cytoprotection sustains ectopic expression of tissue-restricted antigens in the thymus during murine acute GVHD. Blood, 122 (5), pp. 837-841. | Show Abstract | Read more

Development of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEC(high)) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire(+)mTEC(high) to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire(+)mTEC(high) cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire(+)mTEC(high), with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.

Zuklys S, Mayer CE, Zhanybekova S, Stefanski HE, Nusspaumer G, Gill J, Barthlott T, Chappaz S, Nitta T, Dooley J et al. 2012. MicroRNAs control the maintenance of thymic epithelia and their competence for T lineage commitment and thymocyte selection. J Immunol, 189 (8), pp. 3894-3904. | Show Abstract | Read more

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

Lopez-Rios J, Speziale D, Robay D, Scotti M, Osterwalder M, Nusspaumer G, Galli A, Holländer GA, Kmita M, Zeller R. 2012. GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis. Dev Cell, 22 (4), pp. 837-848. | Show Abstract | Read more

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

Pedraza-Alva G, Mérida LB, del Rio R, Fierro NA, Cruz-Muñoz ME, Olivares N, Melchy E, Igras V, Holländer GA, Burakoff SJ, Rosenstein Y. 2011. CD43 regulates the threshold for T cell activation by targeting Cbl functions. IUBMB Life, 63 (10), pp. 940-948. | Show Abstract | Read more

T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.

Krenger W, Blazar BR, Holländer GA. 2011. Thymic T-cell development in allogeneic stem cell transplantation. Blood, 117 (25), pp. 6768-6776. | Show Abstract | Read more

Cytoreductive conditioning regimens used in the context of allogeneic hematopoietic cell transplantation (HCT) elicit deficits in innate and adaptive immunity, which predispose patients to infections. As such, transplantation outcomes depend vitally on the successful reconstruction of immune competence. Restoration of a normal peripheral T-cell pool after HCT is a slow process that requires the de novo production of naive T cells in a functionally competent thymus. However, there are several challenges to this regenerative process. Most notably, advanced age, the cytotoxic pretransplantation conditioning, and posttransplantation alloreactivity are risk factors for T-cell immune deficiency as they independently interfere with normal thymus function. Here, we discuss preclinical allogeneic HCT models and clinical observations that have contributed to a better understanding of the transplant-related thymic dysfunction. The identification of the cellular and molecular mechanisms that control regular thymopoiesis but are altered in HCT patients is expected to provide the basis for new therapies that improve the regeneration of the adaptive immune system, especially with functionally competent, naive T cells.

Lei Y, Ripen AM, Ishimaru N, Ohigashi I, Nagasawa T, Jeker LT, Bösl MR, Holländer GA, Hayashi Y, Malefyt RDW et al. 2011. Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development. J Exp Med, 208 (2), pp. 383-394. | Show Abstract | Read more

Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.

Papadopoulou AS, Dooley J, Linterman MA, Pierson W, Ucar O, Kyewski B, Zuklys S, Hollander GA, Matthys P, Gray DHD et al. 2011. The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor. Nat Immunol, 13 (2), pp. 181-187. | Show Abstract | Read more

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

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