Eur. J. zyxwvutsrqponmlkjihg Immunol. 1987.17: 1265-1269 T cell receptor gene regulation in immature T cells 1265 Christine Kinnono+, Kathleen L. McGuire‘ and Ellen V. Rothenberg Differential regulation of T cell receptor gamma genes in immature thymocyte populations* Division of Biology, California Institute of Technology, Pasadena Immature thymocytes that lack both Lyt-2 (CD8) and L3T4 (CD4) expression can respond rapidly to stimulation with phorbol ester and calcium ionophore by express- ing some gene products characteristic of mature, activated T cells. Here we studied the effect of such short-term stimulation on the number of copies per cell of RNA for components of the T cell receptor complex. Although, upon stimulation, mRNAs for T cell receptor zyxwvut fi chain accumulated to higher levels, the cells did not rapidly increase their expression of a-chain transcripts from rearranged or germ-line genes. Tran- scripts from the C,l (Cy13.4) and C,2 (Cy10.5) genes were differentially regulated. The rarer C,1 transcripts were strongly induced, while the initially abundant C,2 transcripts showed a modest decrease in transcripts per cell within 24 h. Thus, the ratio of these two transcripts could be shifted dramatically prior to any significant change in the cellular composition of the population. These results suggest regulatory processes that may contribute to the observed expression of zyx y products in zyx vitro or in normal development 1 Introduction Precursor of all classes of thymic lymphocytes, and thus of peripheral T cells, can be found in a distinct subpopulation of cells that is retained in the mouse thymus through early adult- hood [I, 21. This population is termed “double negative” because it lacks both the Lyt-2 (CDS) and the L3T4 (CD4) glycoproteins that characteristically mark two major classes of mature T cells. This population is also immature in several molecular and functional respects. In particular, cells in the double-negative population generally do not express mRNA for the zyxwvutsrqpon a chain of the T cell receptor for antigen, although most of them contain transcripts from rearranged p and y- chain loci, a pattern of expression which is also characteristic of immature day 14-17 fetal thymocytes [3-91. This implies that most double-negative cells do not express a classical a/p heterodimeric T cell receptor on their surfaces. However, these cells can make certain functional responses to stimula- tion by calcium ionophores and phorbol esters, which bypass the requirement for membrane receptors [lo]. Among these responses are the synthesis and secretion of the T cell growth factor interleukin 2 (IL2) [ll], with subsequent IL2-depen- dent growth [ll-141. Recent work from several laboratories has focused on the expression of y chain RNA by these cells, in some cases as a definition of the immature cell phenotype, and in others as a means of identifying receptor structures for a distinct T cell lineage. The y chain mRNAs expressed, however, represent products of distinct transcription units, and the y protein observed to date appears to be encoded by only one of these [I 62131 * This work was supported by grants #AI-19752 and CA-39605 from + Upjohn Fellow of the Life Sciences Research Foundation. ’ Supported by NCI felloswhip #CA 07876. Correspondence: Christine Kinnon, Department of Immunology, Institute of Child Health, University of London, 30 Guilford Street, London WClN lEH, GB Abbreviations: cRNA: Complementary RNA IL 2: Interleukin 2 TPA: 12-0-Tetradecanoylphorbol 13-acetate the National Institutes of Health. classes of mRNA. This raises the question of whether different y gene transcripts are coregulated in development. Since dou- ble-negative cells can rapidly alter their expression of growth control genes upon stimulation, we decided to investigate the plasticity of their T cell receptor gene expression under similar conditions. Our results show a striking difference in the regu- lation of two y chain transcription units in these immature cells. 2 Materials and methods 2.1 Animals C57BL/6 mice were bred and maintained in the animal facility at the California Institute of Technology. Mice 4-6 weeks of age were used in all experiments. 2.2 Preparation of double-negative thymocytes Double-negative cells were isolated as previously described [5, 15, 161. Recoveries of double-negative cells were of the order of 1-3% of initial input and < 5% of the cells were judged by flow microfluorimetry to be either CD4’ or CDS’, with ex- pression of IL2 receptors on -70% of the cells. Cells were prepared for flow cytometric analysis by treatment with anti- Lyt-2, anti-L3T4 and anti-IL 2 receptor antibodies as described [15, 161, followed by FITC-conjugated mouse anti- rat immunoglobulin (Cappel Labs., Downington, PA). Flow cytometry was carried out using an Ortho System (Raritan, NJ) 50H Cytofluorograf with a 5-W argon laser (Coherent, Palo Alto, CA). 2.3 RNA isolation Cytoplasmic RNA was prepared as described [17] from freshly isolated double-negative or total thymocytes, or from cells cul- tured for various lengths of time in RPMI medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum, 2 mM L-glutamine, 50 WM 2-mercaptoethanol and 100 unitdm1 penicillin and 0.1 mgtml streptomycin, with or without the addition of 0.15 pg/ml A23187 (Sigma, St. Louis, MO) and 10 ng/ml 12-0-tetradecanoyl phorbol 13-acetate (TPA; Sigma). 0 VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1987 001 4-298018710909- 1265$02.50/0