FINITE LIFESPANS OF T CELL CLONES DERIVED FROM CD34  HUMAN HAEMATOPOIETIC STEM CELLS IN VITRO GRAHAM PAWELEC, 1,2 ROBERT M¨ ULLER, 2 ARNIKA REHBEIN, 1 KARIN H¨ AHNEL 1 and BENEDIKT L. ZIEGLER 2 1 Tu ¨bingen Ageing and Tumour Immunology Group (TATI), Section for Transplantation Immunology and Immunohaematology, 2 Department of Hematology, Oncology, Immunology and Rheumatology, Tu ¨bingen University Medical School, Tu ¨bingen, Germany Abstract—Several studies have documented finite lifespans of at least the vast majority of cultured human T cell lines and clones. However, there is a great deal of variation among the different preparations, ranging from 25 PD up to 100 PD. The cultured T cells in all these studies originated from mature T cells isolated from peripheral blood of adult donors. It was, therefore, impossible to assess the contribution of differences in in vivo age to the subsequent differences between clones in in vitro aging. In an attempt to circumvent this difficulty, we have developed a culture system that supports the differentiation of highly purified human CD34 + cells into CD3 + T cells in vitro. This features the use of a serum-free medium supplemented with the cytokines flt-3 ligand, IL 3, stem cell factor (c-kit ligand) and IL 2, together with IL 7 or oncostatin M (OM). In this way it is possible to perform “longitudinal” studies on T cells derived de novo in vitro. We show here that T cell clones derived under these circumstances also manifest variable finite life expectancies, for which the only uncontrolled (nonstochastic) effects of aging must already have occurred at the stem cell level. © 1999 Elsevier Science Inc. All rights reserved. Key Words: Replicative senescence of T cells, extrathymic T cell differentiation, CD34  stem cells, T cell clones, culture lifespans INTRODUCTION HUMAN T CELL CLONES can be readily generated from the peripheral blood of healthy donors using relatively simple techniques that have remained basically similar for nearly two decades (Bach et al., 1979; Pawelec and Wernet, 1980). Like other somatic cells, T cell clones are commonly found to have finite lifespans, although these vary greatly from clone to clone and from study Correspondence to: Graham Pawelec, Abteilung Innere Medizin II, Medizinische Universita ¨tsklinik und Poliklinik, Otfried-Mu ¨ller-Str. 10, D-72076 Tu ¨bingen, Germany; Tel: +49-7071-298-2805; Fax: +49-7071-294464; E-mail: graham.pawelec@med.uni-tuebingen.de (Received 3 July 1998; Accepted 27 July 1998) Experimental Gerontology, Vol. 34, No. 1, 69 –77, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0531-5565/99 $–see front matter PII S0531-5565(98)00049-7 69