Distinct transcriptional programs in thymocytes responding to T cell receptor, Notch, and positive selection signals

Abstract

T cell antigen receptor (TCR) signaling is necessary but not sufficient to promote the positive selection of CD4+CD8+ thymocytes into CD4+ or CD8+ mature T cells. Notch signaling has also been implicated as a potential regulator of both CD4/CD8 T cell development and TCR signaling. However, the relationship between positive selection, TCR signaling, and Notch remains unclear. Here we use DNA microarray analysis to compare gene expression changes in CD4+CD8+ double-positive thymocytes undergoing positive selection, TCR stimulation, and Notch activation. We find that the genes induced during positive selection can be resolved into two distinct sets. One set, which we term "TCR-induced," is also induced by in vitro TCR stimulation and contains a large proportion of transcription factors. A second set, which we term "positive-selection-induced," is not induced by in vitro TCR simulation and contains a large proportion of genes involved in signal transduction pathways. Genes induced by Notch activity overlap substantially with genes induced during positive selection. We also find that Notch activity potentiates the effects of TCR stimulation on gene expression. These results help to identify TCR- and positive-selection-specific transcriptional events and help to clarify the relationship between positive selection and Notch.

Fig. 1.

Gene expression differences in thymocytes in response to positive selection, Notch activity, or TCR stimulation. Whole wild-type B6 thymocytes, MHC^-/- thymocytes stimulated through TCR, or CD4⁺CD8⁺ (DP) thymocytes from 5CC7, AND, F5, or P14 TCR or NotchIC tg mice were compared with a reference population with microarray slides spotted with ≈2,700 immunologically important probes. Sorted DP MHC^-/- thymocytes served as reference for all DP test populations. Unfractionated MHC^-/- thymocytes served as reference for all other test populations, unless specified otherwise. One hundred seventy-seven genes were identified to have statistically significant expression changes and clustered by using cluster 2.12. Rows correspond to individual genes, and columns correspond to test thymocyte populations. Genes with similar expression patterns cluster together, and the tree diagram displayed on the left represents the relative similarity in expression pattern between genes. Four major expression clusters were apparent: genes induced during selection (1a and 1b), induced by TCR stimulation (2), repressed by TCR stimulation (3a, 3b, and 3c), and induced by NotchIC activity (4). Gene identities including unigene v.116 numbers are listed with the following functional identifiers: st, signal transduction (purple type), tf, transcription factor (blue type); cc, cell cycle (orange type). Gene expression changes for lymph node T cells in vitro TCR-stimulated for 8 h compared with unstimulated T cells are displayed alongside clusters 1 and 2. Genes marked with ^* had low hybridization signal or high covariance in some thymocyte populations. Confirmation of individual expression patterns should be performed before further investigating individual genes. For data values, see Tables 2 and 3, which are published as supporting information on the PNAS web site.

Fig. 2.

Effect of Notch activity on TCR-driven gene expression changes. (A) Microarray analysis of MHC^-/- thymocytes stimulated with plate-bound anti-TCR for 2, 4, 8, and 16 h. Ninety TCR-responsive genes were clustered as in Fig. 1. Four categories were apparent: rapidly induced or repressed and gradually induced (represented by two independent clusters) or repressed genes. (B) Microarray and flow cytometric analysis of CD8 and CD69 expression on TCR-stimulated MHC^-/- thymocytes. (C) Effect of Notch activity on TCR-dependent rapidly and gradually modulated genes was determined by microarray comparison of normal littermate control MHC^-/- and NotchIC tg MHC^-/- thymocytes stimulated through TCR for 2, 4, 8, or 16 h. Graphs of representative genes of rapid (Upper) and gradual (Lower) inducers and repressors are displayed.