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*Ludwig Institute for Cancer Research Ltd., Lausanne Branch, 1066 Epalinges, Switzerland;
Ecole Polytechnique Fédèrale de Lausanne, Swiss Institute for Experimental Cancer Research, School of Life Sciences, 1015 Lausanne, Switzerland;
WHO Immunology Research and Training Center and
Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland; and
¶Centre d’Immunologie de Marseille-Luminy, Marseille, France
It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4–CD8– thymocytes. Notch1 is progressively down-regulated at the CD4+CD8+ and mature CD4+ or CD8+ thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1+CD25– cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117+ early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Swiss National Science Foundation PP00A-116896 (to S.A.L.), 3100A0-119725 (to F.R.), 320000-116197 (to F.T.C.), and 3100A-120165 (to H.R.M.).
2 E.M. and A.W. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research Ltd., Lausanne Branch, chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address: HughRobson.MacDonald{at}licr.unil.ch
4 Abbreviations used in this paper: N1, Notch1; DL, Delta-like; LCMV, lymphocytic choriomeningitis virus; BM, bone marrow; DN, double negative; ISP, immature single positive; iIEL, intestinal intraepithelial lymphocytes; DP, double positive; CLP, common lymphoid precursor; K5, keratin 5; DAPI, 4',6-diamidino-2-phenylindole; EGF, epidermal growth factor-like.
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