RT Journal Article T1 Eph/ephrin-B-mediated cell-to-cell interactions govern MTS20+ thymic epithelial cell development A1 Montero Herradon, Sara A1 Garcia-Ceca Hernández, Javier A1 Sánchez del Collado, Beatriz A1 Zapata González, Agustín AB Thymus development is a complex process in which cell-to-cell interactions between thymocytes and thymic epithelial cells (TECs) are essential to allow a proper maturation of both thymic cell components. Although signals that control thymocyte development are well known, mechanisms governing TEC maturation are poorly understood, especially those that regulate the maturation of immature TEC populations during early fetal thymus development. In this study, we show that EphB2-deficient, EphB2LacZ and EphB3-deficient fetal thymuses present a lower number of cells and delayed maturation of DN cell subsets compared to WT values. Moreover, deficits in the production of chemokines, known to be involved in the lymphoid seeding into the thymus, contribute in decreased proportions of intrathymic T cell progenitors (PIRA/B+) in the mutant thymuses from early stages of development. These features correlate with increased proportions of MTS20+ cells but fewer MTS20− cells from E13.5 onward in the deficient thymuses, suggesting a delayed development of the first epithelial cells. In addition, in vitro the lack of thymocytes or the blockade of Eph/ephrin-B-mediated cell-to-cell nteractions between either thymocytes–TECs or TECs–TECs in E13.5 fetal thymic lobes coursed with increased proportions of MTS20+ TECs. This confirms, for the first time, that the presence of CD45+ cells, corresponding at these stages to DN1 and DN2 cells, and Eph/ephrin-B-mediated heterotypic or homotypic cell interactions between thymocytes and TECs, or between TECs and themselves, contribute to the early maturation of MTS20+ TECs. SN 0948-6143 (Print) 1432-119X (Online) YR 2016 FD 2016-08 LK https://hdl.handle.net/20.500.14352/17614 UL https://hdl.handle.net/20.500.14352/17614 LA eng NO Aasheim HC, Delabie J, Finne EF (2005) Ephrin-A1 binding toCD4+ T lymphocytes stimulates migration and induces tyrosinephosphorylation of PYK2. Blood 105:2869–2876. doi:10.1182/blood-2004-08-2981Alfaro D et al (2007) EphrinB1-EphB signaling regulates thymocyteepithelium interactions involved in functional T cell development.Eur J Immunol 37:2596–2605.doi:10.1002/eji.200737097Alfaro D, Munoz JJ, Garcia-Ceca J, Cejalvo T, Jimenez E, Zapata A (2008) Alterations in the thymocyte phenotype of EphB-deficient Histochem Cell Biol (2016) 146:167–182 1811 3 mice largely affect the double negative cell compartment. Immunology 125:131–143. doi:10.1111/j.1365-2567.2008.02828.xAlfaro D, Munoz JJ, Garcia-Ceca J, Cejalvo T, Jimenez E, Zapata AG (2011) The Eph/ephrinB signal balance determines the pattern of T-cell maturation in the thymus. Immunol Cell Biol 89:844–852.doi:10.1038/icb.2010.172Alfaro D et al (2015) EphB2 and EphB3 play an important role in the lymphoid seeding of murine adult thymus. J Leukoc Biol 98:883–896. doi:10.1189/jlb.1HI1114-568RAnderson G, Jenkinson EJ (2001) Lymphostromal interactions in thymic development and function. Nat Rev Immunol 1:31–40.doi:10.1038/35095500Anderson G, Jenkinson EJ, Rodewald HR (2009) A roadmap forthymic epithelial cell development. Eur J Immunol 39:1694–1699. doi:10.1002/eji.200939379Bennett AR, Farley A, Blair NF, Gordon J, Sharp L, Blackburn CC (2002) Identification and characterization of thymic epithelial progenitor cells. Immunity 16:803–814Blackburn CC, Manley NR (2004) Developing a new paradigmfor thymus organogenesis. Nat Rev Immunol 4:278–289.doi:10.1038/nri1331Blackburn CC et al (1996) The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors. Proc Natl Acad Sci USA 93:5742–5746Boehm T, Scheu S, Pfeffer K, Bleul CC (2003) Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTbetaR. J Exp Med 198:757–769. doi:10.1084/jem.20030794Calderon L, Boehm T (2011) Three chemokine receptors cooperatively regulate homing of hematopoietic progenitors to the embryonic mouse thymus. Proc Natl Acad Sci USA 108:7517–7522. doi:10.1073/pnas.1016428108Colucci F, Soudais C, Rosmaraki E, Vanes L, Tybulewicz VL, Di Santo JP (1999) Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation. J Immunol 162:2761–2765Davy A, Soriano P (2005) Ephrin signaling in vivo: look both ways.Dev Dyn 232:1–10. doi:10.1002/dvdy.20200Depreter MG et al (2008) Identification of Plet-1 as a specific marker of early thymic epithelial progenitor cells. Proc Natl Acad Sci USA 105:961–966.doi:10.1073/pnas.0711170105Garcia-Ceca J et al (2009) On the role of Eph signalling in thymus histogenesis; EphB2/B3 and the organizing of the thymic epithelial network. Int J Dev Biol 53:971–982. doi:10.1387/ijdb.082702jgGarcia-Ceca J, Alfaro D, Montero-Herradon S, Zapata AG (2013) Eph/ephrinB signalling is involved in the survival of thymic epithelial cells. Immunol Cell Biol 91:130–138. doi:10.1038/icb.2012.59Garcia-Ceca J, Alfaro D, Montero-Herradon S, Tobajas E, Munoz JJ,Zapata AG (2015) Eph/ephrins-mediated thymocyte-thymic epithelial cell interactions control numerous processes of thymus biology. Front Immunol 6:333. doi:10.3389/fimmu.2015.00333Gill J, Malin M, Hollander GA, Boyd R (2002) Generation of a complete thymic microenvironment by MTS24(+) thymic epithelial cells. Nat Immunol 3:635–642. doi:10.1038/ni812Godfrey DI, Izon DJ, Tucek CL, Wilson TJ, Boyd RL (1990) The phenotypic heterogeneity of mouse thymic stromal cells. Immunology70:66–74Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder J, Pawson T, Klein R (1996) Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 86:35–46Hjorthaug HS, Aasheim HC (2007) Ephrin-A1 stimulates migration of CD8+ CCR7+ T lymphocytes. Eur J Immunol 37:2326–2336. doi:10.1002/eji.200737111Hollander GA et al (1995) Developmental control point in induction of thymic cortex regulated by a subpopulation of prothymocytes.Nature 373:350–353. doi:10.1038/373350a0Itoi M, Kawamoto H, Katsura Y, Amagai T (2001) Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage. Int Immunol 13:1203–1211Jenkinson WE, Rossi SW, Parnell SM, Agace WW, Takahama Y,Jenkinson EJ, Anderson G (2007) Chemokine receptor expression defines heterogeneity in the earliest thymic migrants. Eur J Immunol 37:2090–2096. doi:10.1002/eji.200737212Jotereau F, Heuze F, Salomon-Vie V, Gascan H (1987) Cell kinetics in the fetal mouse thymus: precursor cell input, proliferation, and emigration. J Immunol 138:1026–1030Kitamura T et al (2008) Enhancement of lymphocyte migration and cytokine production by ephrinB1 system in rheumatoid arthritis. Am J Physiol Cell Physiol 294:C189–C196. doi:10.1152/ajpcell.00314.2007Klug DB, Carter C, Crouch E, Roop D, Conti CJ, Richie ER (1998) Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment. Proc Natl Acad Sci USA 95:11822–11827Klug DB, Carter C, Gimenez-Conti IB, Richie ER (2002) Cuttingedge: thymocyte-independent and thymocyte-dependentphases of epithelial patterning in the fetal thymus. J Immunol 169:2842–2845Knoll B, Drescher U (2002) Ephrin-As as receptors in topographic projections. Trends Neurosci 25:145–149Laurent J, Bosco N, Marche PN, Ceredig R (2004) New insights into the proliferation and differentiation of early mouse thymocytes.Int Immunol 16:1069–1080. doi:10.1093/intimm/dxh108Liu C et al (2005) The role of CCL21 in recruitment of T-precursor cells to fetal thymi. Blood 105:31–39. doi:10.1182/blood-2004-04-1369Liu C et al (2006) Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.Blood 108:2531–2539. doi:10.1182/blood-2006-05-024190Munoz JJ, Cejalvo T, Alonso-Colmenar LM, Alfaro D, Garcia-Ceca J, Zapata A (2011) Eph/ephrin-mediated interactions in the thymus.NeuroImmunoModulation 18:271–280. doi:10.1159/000329490Pasquale EB (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133:38–52.doi:10.1016/j.cell.2008.03.011Perez White BE, Getsios S (2014) Eph receptor and ephrin function in breast, gut, and skin epithelia. Cell Adhes Migr 8:327–338. doi:10.4161/19336918.2014.970012Romano R, Palamaro L, Fusco A, Giardino G, Gallo V, Del Vecchio L, Pignata C (2013) FOXN1: a master regulator gene of thymic epithelial development program. Front Immunol 4:187.doi:10.3389/fimmu.2013.00187Rossi SW, Jenkinson WE, Anderson G, Jenkinson EJ (2006) Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium. Nature 441:988–991. doi:10.1038/nature04813Rossi SW et al (2007a) Redefining epithelial progenitor potential in the developing thymus. Eur J Immunol 37:2411–2418.doi:10.1002/eji.200737275Rossi SW et al (2007b) RANK signals from CD4(+)3(−) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla. J Exp Med 204:1267–1272. doi:10.1084/jem.20062497Shakib S, Desanti GE, Jenkinson WE, Parnell SM, Jenkinson EJ,Anderson G (2009) Checkpoints in the development of thymic cortical epithelial cells. J Immunol 182:130–137Sharfe N, Freywald A, Toro A, Dadi H, Roifman C (2002) Ephrin stimulationmodulates T cell chemotaxis. Eur J Immunol 32:3745–3755. doi:10.1002/1521-4141(200212)32:12<3745::AIDIMMU3745>3.0.CO;2-MStimamiglio MA et al (2010) EphB2-mediated interactions are essential for proper migration of T cell progenitors during fetal thymus colonization. J Leukoc Biol 88:483–494. doi:10.1189/jlb.0210079Trinidad EM, Ballesteros M, Zuloaga J, Zapata A, Alonso-Colmenar LM (2009) An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression. Blood 114:5081–5090. doi:10.1182/blood-2009-03-210617van Ewijk W, Shores EW, Singer A (1994) Crosstalk inthe mouse thymus. Immunol Today 15:214–217.doi:10.1016/0167-5699(94)90246-1van Ewijk W, Hollander G, Terhorst C, Wang B (2000) Stepwise development of thymic microenvironments in vivo is regulated by thymocyte subsets. Development 127:1583–1591Wang JH, Nichogiannopoulou A, Wu L, Sun L, Sharpe AH, Bigby M, Georgopoulos K (1996) Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5:537–549Wilkinson B, Owen JJ, Jenkinson EJ (1999) Factors regulating stem cell recruitment to the fetal thymus. J Immunol 162:3873–3881Zamora DO, Babra B, Pan Y, Planck SR, Rosenbaum JT (2006)Human leukocytes express ephrinB2 which activates microvascular endothelial cells. Cell Immunol 242:99–109. doi:10.1016/j.cellimm.2006.10.001Zhu M, Fu YX (2008) Coordinating development of medullarythymic epithelial cells. Immunity 29:386–88.doi:10.1016/j.immuni.2008.09.001Zlotoff DA, Bhandoola A (2011) Hematopoietic progenitor migration to the adult thymus. Ann N Y Acad Sci 1217:122–138.doi:10.1111/j.1749-6632.2010.05881.x NO Ministerio de Ciencia e Innovación (España) NO Ministerio de Economía y Competitividad (España) NO Ministerio de Sanidad y Consumo (España) DS Docta Complutense RD 4 may 2024