In this article, we report a large-scale functional genetic screen for inhibitors of TCR signaling. We isolated many known signaling molecules - such as Lck, ZAP70, Syk, PLCγ1 - as novel truncation mutants (probably created during library preparation) with dominant-negative effects. In addition, we also discovered molecules previously unknown to this pathway, including transmembrane molecules (EDG-1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK-2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin; see Table 1). Of note, we also identified a novel molecule, TRAC-1, which had lymphoid and hematopoietic specific expression (Figure 5b).
We showed that EDG1, PAK2, and Grb7, genes originally described in different contexts, are also expressed in lymphocytes (Figure 5). This is not unexpected, since the retroviral cDNA libraries were generated using mRNA from human lymphoid organs such as thymus, spleen, lymph nodes and bone marrow. Our expression data are generally consistent with those published by other investigators. For example, EDG1 was reported to be expressed in human natural killer cells  and dendritic cells . PAK2 is expressed ubiquitously in human tissues  and in Jurkat cells . Grb7 has a broad expression in human (pancreas, placenta, kidney, prostate and small intestines) . Grb7 was not easily detectable by northern blot in thymus, spleen and PBL, but its expression was detected in specific lymphocyte subsets (Figure 5e). This indicates that our screen is capable of identifying genes with potentially important roles in lymphocyte activation whose expression is not limited to the lymphoid system. The fact that these genes' expression is not limited to the lymphoid system does not diminish the potential role they could play in lymphocyte activation. For example, the Ras-Raf-MAP kinase pathway is ubiquitously present in many tissues and cell types, as well as conserved evolutionarily, but this pathway has also been shown to be important in lymphocyte signaling.
In the 'post-genomics' era, the novelty of discovery lies in assigning novel functions to gene products. In our screens, for example, we identified two hits representing cytoplasmic truncated versions of EDG-1, a receptor for S1P . Interestingly, FTY720, a potent immunosuppressant in advanced clinical development, has been shown to act through EDG-1 and S1P signaling pathways [35, 36]. The fact that truncated EDG-1 proteins were identified in our T-cell activation screen suggests potential intersections of the TCR signaling pathway and the S1P signaling pathway, as well as new insights into the mechanisms of action of FTY720.
Our results also call for attention to potential differences between related family members. For example, PAK-1 (instead of PAK-2), c-Raf-1 (instead of A-Raf-1) and Grb2 (instead of Grb7) have been reported to be associated with the TCR signal transduction pathway [25, 37–40]. Our functional genetic screens identified PAK2, A-Raf-1, and Grb7 as important regulators of TCR-induced CD69 expression. It is possible that the dominant-negative proteins we cloned also inhibit other related family members. Alternatively, it is equally possible that the previously reported dominant-negative forms of PAK1, c-Raf-1, and to a lesser extent, Grb2, may have inhibited PAK2, A-Raf-1 and Grb7, respectively. In fact, binding of the human immunodeficiency virus (HIV) Nef protein and subsequent activation of the PAK-related kinase and phosphorylation of its substrate can be readily detected in both infected primary T lymphocytes and macrophages . When the HIV-Nef-associated kinase was characterized carefully, it became clear that this kinase was PAK2 and not PAK1 [42, 43]. This example supports the notion that PAK2 could be the more relevant kinase in T-cell signaling. Of course, it is entirely possible that these related family members are not mutually exclusive in participating in the TCR signal-transduction pathway.
In conclusion, we have demonstrated a successful approach for discovering and validating, in a functionally relevant context, important immune regulators on a genome-wide scale. This approach provides a tool for functional cloning of regulators in numerous signal-transduction pathways [44, 45]. For example, B-cell activation-induced CD69 expression  and, recently, the IL-4-induced immunoglobulin E class switch , have also been shown to be amenable to genetic perturbation following introduction of retroviral cDNA or random cyclic peptide libraries. Importantly, the outlined strategy, which requires no prior sequence information of the players involved, does not bias the search to previously known signaling molecules, molecules flagged by DNA-array technologies, or signaling molecules discovered in other contexts. This approach has added to the list of potential players in T-cell biology that have not been identified in other standard pathway-mapping techniques.