GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.

Activation of mitogen-activated protein kinase (MAPK) pathways leads to cellular differentiation and/or proliferation in a wide variety of cell types, including developing thymocytes. The basic helix-loop-helix (bHLH) proteins E12 and E47 and an inhibitor HLH protein, Id3, play key roles in thymocyte differentiation. We show here that E2A DNA binding is lowered in primary immature thymocytes consequent to T cell receptor (TCR)-mediated ligation. Whereas expression of E2A mRNA and protein are unaltered, Id3 transcripts are rapidly induced upon signaling from the TCR. Activation of Id3 transcription is regulated in a dose-dependent manner by the extracellular signal-regulated kinase (ERK) MAPK module. These observations directly connect the ERK MAPK cascade and HLH proteins in a linear pathway.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant‐negative p21ras protein (H‐rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck‐H‐rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR‐derived signals in mature CD4+8‐ or CD8+4‐ thymocytes. In contrast, some TCR‐derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant‐negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H‐Y‐specific TCR and lck‐H‐rasN17 demonstrated that antigen‐specific negative selection occurs normally in the presence of p21H‐rasN17. Superantigen‐induced negative selection in vivo also proceeded unhindered in H‐rasN17 thymocytes. In contrast, positive selection of thymocytes in the H‐Y mice was severely compromised by the presence of p21H‐rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.

SummaryDuring T cell development, interaction of the T cell receptor (TCR) with cognate ligands in the thymus may result in either maturation (positive selection) or death (negative selection). The intracellular pathways that control these opposed outcomes are not well characterized. We have generated mice expressing dominant-negative P.as (dnRas) and Mek-1 (dMek) transgenes simultaneously, either in otherwise normal animals, or in animals expressing a transgenic TCR, thereby permitting a comprehensive analysis of peptide-specific selection. In this system, thymocyte maturation beyond the CD4+8 + stage is blocked almost completely, whereas negative selection, assessed using an in vitro deletion protocol, is quantitatively intact. This suggests that activation of the mitogen-activated protein kinase (MAPK) cascade is necessary for positive selection, but irrelevant for negative selection. Generation of~/~ and of CD4-8-tx/13 T cells proceeds normally despite blockade of the MAPK cascade. Hence, only cells that mature via conventional, TCP.-mediated repertoire selection require activation of the MAPK pathway to complete their maturation.