EGR-1 is rapidly up-regulated after TCR stimulation, and this induction is dependent on Ras / MAPK activation. In MHC-deficient thymi, EGR-1 expression is dram atically reduced, b u t can be induced in vitro by anti-CD3e antibody treatm ent (Shao et al., 1997). Transgenic mice over-expressing EGR-1 w ere able to positively select CD8+ SP cells on a non-selecting background and even on a MHC-deficient background (Miyazaki and Lemonnier, 1998). A lthough these data imply a role for EGR-1 in positive selection of CD8+ SP cells, recent reports (using pharmacological inhibition of EGR-1) have suggested that its expression is not required for CD8 lineage com m itm ent and selection (Basson et al., 2000). In sum m ary, EGR-1 is one of the earliest dow nstream targets of TCR signalling, and thus m ay potentially regulate gene transcription during selection, b u t does not seem to be essential for the selection process itself.
Nurr77, a m em ber of the nuclear orphan receptor family of TFs, appears to be involved in negative selection. Three lines of evidence support this role for Nurr77: its activity in TCR-induced apoptosis (Liu et al., 1994; Woronicz et al., 1994), the fact that a dom inant negative isoform blocks negative selection (Lee et al., 1995; Z hou et al., 1996), and the observation that a constitutively active version of the molecule induces apoptosis (Lee et al., 1995). How ever, Nurr77*'^' mice show no gross selection abnormalities, suggesting that redundant factors (possibly other nuclear orphan receptors, such as NOR-1) m ay function in these processes.
IRF-1 (interferon regulatory factor 1) appears to play a role in both positive and negative selection. IRF-1'^‘ mice have reduced num bers of thymic and peripheral CD8+ T cells, and positive selection of MHC class I -restricted transgenes is im paired on an IRF-1'^' background (Penninger et al., 1997). This is not due to a thymic strom a defect, as norm al developm ent of WT thymocytes can be supported by IRF-l-deficient stroma, both in FTOC and in bone m arrow chimeras. In terms of negative selection, a 1,000-fold increase in the am ount of selecting peptide was required to delete TCR transgenic thymocytes on an IFR-l"'^' background (Penninger et al., 1997). IRF-1 only seems to play a role in late T cell developm ent, as its expression is not detectable im m ature thymocytes before TCRap expression.
One of the crucial functions of TCR signalling is the induction of expression of cytokines, especially IL-2, which is vital for T cell physiology. IL-2 expression is regulated by several transcription factors that are dow nstream effectors of TCR signalling cascades, namely NFAT, NF-kB and CREB.
NFAT (nuclear factor of activated T cells) was initially identified as an inducible protein complex that could bind a regulatory elem ent in the IL-2 prom oter, b u t in fact NFAT binding sites are also present in the IL-3, IL-4 and TN F-a prom oters. TCR signalling activates NFAT via the PLC-y/IPg/Ca^^/calcineurin pathw ay. Calcineurin dephosphorylates inactive NFAT in the cytosol, thereby unm asking a nuclear localisation signal. This change leads to rapid translocation to the nucleus, w here NFAT pairs w ith A PI complexes composed of Fos and Jun dimers (reviewed in Kuo, 1999). Over-expression of Vav also leads to a m arked increase in NFAT activity and IL-2 expression (Wu et al., 1995). Im portantly, NFAT-4'^" mice show a
reduction in SP cells (Oukka et al., 1998). This defect seems to be due to increased sensitivity of DP thymocytes to apoptosis, as the expression of survival gene Bcl-2 is reduced.
NF-KB transcription factors include NF-KB-1/-2, Rel-A/-B and c-Rel. Their activity is controlled at the post-translational level by association w ith inhibitory IkB proteins. TCR signalling induces degradation of IKB, which releases NFkB and allows it to translocate to the nucleus. A lthough NFKB-1"^' mice show ed no abnorm ality in T cell developm ent, this could be due to functional redundancy betw een the several NFkB proteins (Sha et al., 1995; Snapper et al., 1996). To circumvent this, transgenic mice expressing a '"super-inhibitory" form of IKB under the control of T cell specific prom oters w ere generated (Boothby et al., 1997; Esslinger et al., 1997). Since this m utant IkB cannot be phosphorylated and degraded in response to TCR engagem ent, the function of all NFkB proteins are inhibited. These mice displayed significantly decreased num bers of peripheral CDS T cells, and a severe proliferative defect in response to TCR cross-linking (am ong other activation defects).
CREB is a basic/leucine zipper TF that binds CRE sequences present in regulatory regions of m any genes, including TCRa, TCRP, CD35 and CDSa (see 2.4.4). CREB is responsive to both TCR-dependent and TCR-independent signals. Am ong the latter, CREB activity is particularly sensitive to variations in intracellular cyclic-AMP levels. Treatm ent of foetal lobes with cyclic-AMP analogues, in FTOC, resulted in a major loss of DP cells, presum ably due to apoptosis (Lalli et al., 1996). On the other hand, upon TCR engagem ent, CREB is rapidly phosphorylated and activated via a single pathw ay that involves Lck/Ras/R af/M EK /RSK -2 (M uthusam y and Leiden, 1998). Peripheral T cells from transgenic mice expressing a dom inant-negative CREB failed to proliferate efficiently or to produce appropriate cytokine (namely IL-2) responses (Barton et al., 1996). These defects correlated w ith a decreased induction of transcription factors Fos and Jun (API complex). CREB(-) cells underw ent apoptosis in response to a variety of stimuli that activated CREB(+) T cells (Barton et al., 1996).