922 N. zyxwvutsrqpon C. Moore, G. Anderson, C. A. Smith et al. Eur. J. Immunol. 1993.23: 922-927 zy Nel C. Moore, zyxwvutsr Graham Anderson, Christopher A. Smith, John J. T. Owen and Eric J. Jenkinson Department of Anatomy, The Medical School, University of zyxwvuts Birmingham, Birmingham Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction* Using a semi-quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages.The expression of interleukin (1L)-7, stem cell factor (SCF), IL-la and granulocyte-monocyte-colony-stimulating factor (GM-CSF) mRNA was mapped to individual stromal cell types, while the expression of IL-la and GM-CSF, along with interferon (IFN)-y and IL-4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid-specific cytokine genes was selectively down-regulated in thymocytes undergoing maturation. CD3-"O4+8+ cells, representing an interme- diate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFNy mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class 11+ cortical epithelial cells strongly expressed IL-7 and SCF, but only limited expression of IL-la and GM-CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL-la and GM-CSF but not IL-7. The importance of these cytokine profiles in relation to T cell development is discussed. 1 Introduction The differentiation of T cell precursors into immature CD4+8+ cortical thymocytes and, ultimately, into mature single positive CD4+ or CD8+ T cells occurs in the thymus [l]. During this development interaction with cells of the thymic stroma is known to play a vital role in thymocyte maturation and selection [2, 31. Removal of thymocytes from their stromal environment not only perturbs matura- tion [4], but also alters their response to non-physiological stimuli [5], and enhances spontaneous cell death [6]. These factors all point to an important role for stromal cells and/or stromal cell products inT cell development. In this context, a number of cytokines have been implicated in Tcell development [7-lo]. However, many of these studies are based on cultures of isolated thymocytes where interpreta- tion is complicated by the factors mentioned above. On the other hand, there is only limited information available on the association between cytokine gene expression and particular thymocyte subpopulations or stromal cell types zyxwv in vivo. [I 111751 zyxwvu * This work was supported by an MRC programme grant. G.A. holds a Wellcome Prize Studentship. Correspondence: Nel C. Moore, Department of Anatomy, The Medical School, University of Birmingham, Birmingham B15 2?T, GB Abbreviations: dGuo: Deoxyguanosine Fd Fetal day GM- CSF Granulocyte monocyte-CSF PCR. Polymerase chain reac- tion SCF Stem cell factor Key words: Cytokines / Polymerase chain reaction / Stroma / Thymocytes I Thymus To relate changes in cytokine gene expression to normal thymocyte development and to localize expression to particular cell types we have used a semiquantitative polymerase chain reaction (PCR) technique [ll] to study gene expression profiles in individual components of the thymic stroma obtained by immunomagnetic selection, in freshly isolated staged fetal thymocytes and thymocytes at different maturational stages defined by CD4, CD8 and TcR expression. Using this approach we have examined the expression of a number of cytokines in thymus development and shown that the expression of IG7, stem cell factor (SCF), IL-la and granulocyte-monocyte-CSF (GM-CSF) can be mapped to individual stromal cell types. Expression of the genes encoding IL-la and GM-CSF, along with IFN-y and IL-4, can also be demonstrated in the lymphoid compartment of the day 14 fetal thymus but undergo selective down- regulation on further development. 2 Materials and methods 2.1 Animals Timed matings were carried out using BALB/c mice. The day of gestation was calculated by the plug date (= day 0). Thymus lobes were removed on each day of the gestation from fetal day (Fd) 14 to 18, and from new born animals. 2.2 Antibodies For selection the following antibodies were bound onto either anti-rat Ig- or anti-mouse Ig-coated magnetic beads 0014-2980/9310404-0922$3.50 + .2510 zyxwvutsr 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993