The enzymes generated by TCR signaling activate transcription factors that bind to regulatory regions of numerous genes in T cells and thereby enhance transcription of these genes (Fig. 1). Much of our understanding of the transcriptional regulation of genes in T cells is based on analyses of cytokine gene expression. The transcriptional regulation of most cytokine genes in T cells is controlled by the binding of transcription factors to nucleotide sequences in the promoter and enhancer regions of these genes. For instance, the promoter located 5′ of the coding exons of the IL2 gene contains a segment of approximately 300 base pairs that contains binding sites for several different transcription factors. All of these sites must be occupied by transcription factors for maximal expression of the IL2 gene. Different transcription factors are activated by different cytoplasmic signal transduction pathways, and the requirement for multiple transcription fac tors accounts for the need to activate many signaling pathways after antigen recognition. The same principles are true for the induced expression of many genes in T cells, including those encoding cytokine receptors and effector molecules, although different genes may be responsive to different combinations of transcription factors.
Fig1. Activation of transcription factors in T cells. Multiple signaling pathways converge in antigen stimulated T cells to generate transcription factors that stimulate expression of various genes (in this case, the interleukin-2 (IL2) gene). The calcium-calmodulin pathway activates nuclear factor of activated T cells (NFAT), and the RAS and RAC pathways generate the two components of AP-1. Less is known about the link between TCR signals and nuclear factor-κB (NF-κB) activation. (NF-κB is shown as a complex of two subunits, which in T cells are typically the p50 and p65 proteins, named for their molecular sizes in kilodaltons.) Protein kinase C (PKC) is important in T-cell activation, and the PKCθ isoform is particularly important in activating NF-κB. These transcription factors function coordinately to regulate gene expression. Note also that the various signaling pathways are shown as activating unique transcription factors, but there may be considerable overlap, and each pathway may play a role in the activation of multiple transcription factors. ERK, Extracellular receptor activated kinase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; JNK, c-Jun N-terminal kinase.
Three transcription factors that are activated in T cells by anti gen recognition and appear to be critical for most T-cell responses are nuclear factor of activated T cells (NFAT), AP-1, and NF-κB.
• NFAT is a transcription factor required for the expression of genes encoding IL-2, IL-4, TNF, and other cytokines. NFAT is present in an inactive, serine-phosphorylated form in the cytoplasm of resting T lymphocytes. It is activated by the calcium-calmodulin–dependent phosphatase calcineurin. Calcineurin dephosphorylates cytoplasmic NFAT, thereby exposing a nuclear localization signal that permits NFAT to translocate into the nucleus. Once it is in the nucleus, NFAT binds to the regulatory regions of the IL2 and other genes, usually in association with other transcription factors, such as AP-1. The mechanism of activation of NFAT was discovered indirectly by studies of the immunosuppressive drug cyclosporine. This drug and the functionally similar com pound, tacrolimus (FK506), are natural products of fungi and are widely used to prevent and treat transplant rejection. They function largely by blocking T-cell cytokine gene transcription. Cyclosporine binds to a cytosolic protein called cyclophilin, and tacrolimus binds to a protein called FK506-binding protein (FKBP). Cyclosporine cyclophilin complexes and tacrolimus-FKBP complexes bind to and inhibit calcineurin (this is why these drugs are called calcineurin inhibitors) and thereby block translocation of NFAT into the nucleus.
• AP-1 is a transcription factor found in many cell types; it is specifically activated in T lymphocytes by TCR-mediated signals. AP-1 is actually the name for a family of DNA-binding factors composed of dimers of two proteins that bind to one another through a shared structural motif called a leucine zipper. The best characterized AP-1 factor is composed of the proteins FOS and JUN. As discussed previously, the formation of active AP-1 typically involves synthesis of the FOS protein and phosphorylation of preexisting JUN protein, both stimulated by MAP kinases that are activated by TCR-induced signals. AP-1 physically associates with other transcription factors in the nucleus, and it works best in combination with NFAT. Thus, AP-1 activation represents a convergence point of several TCR-initiated signaling pathways.
• NF-κB refers to a group of closely related transcription factors that are activated in response to TCR signals and are essential for cytokine synthesis. NF-κB proteins are homodimers or heterodimers of proteins that are homologous to c-REL and are important in the transcription of many genes in diverse cell types. The NF-κB pathway is important not only for antigen receptor–mediated lymphocyte activation but also for responses to Toll-like receptor and cytokine signaling.
The links between different signaling proteins, activation of transcription factors, and functional responses of T cells are often difficult to establish because there are complex and incompletely understood interactions between signaling pathways. Also, for the sake of simplicity, we often discuss signaling as a set of linear pathways, but we know this does not reflect the more complex and interconnected reality. Finally, we have focused on selected pathways to illustrate how antigen recognition may lead to biochemical alterations, but it is clear that many other signaling molecules are also involved in antigen-induced lymphocyte activation.
An additional mechanism by which T-cell activation is regulated involves microRNAs (miRNAs). miRNAs are small, noncoding RNAs that are transcribed from DNA but are not translated into proteins. The function of miRNAs is to inhibit expression of specific genes. miRNAs are initially generated in the nucleus as longer primary transcripts that are processed by an endoribonuclease called Drosha into shorter pre-miRNAs that have a stem loop structure and can be exported into the cytosol. In the cytosol, pre-miRNAs are processed by another endoribonuclease called Dicer into short, double-stranded miRNAs, 21 to 22 base pairs in length, one strand of which can pair with a complementary sequence in a number of cellular messenger RNAs (mRNAs). These mRNAs associate both with miRNAs and with Argonaute proteins to form complexes known as RISC (RNA-induced silencing complexes). If the 6- to 8-base pair miRNA sequence that recognizes mRNAs (also called the seed sequence) is not perfectly complementary to the mRNA, the mRNA is prevented from being translated efficiently. mRNAs may be targeted for degradation when complementarity is perfect. In either case, the result is a reduction in the abundance of proteins encoded by genes targeted by miRN As. In T cells, the expression of the majority of miRNAs is globally reduced upon activation. In addition, the Argonaute protein is ubiquitinated and degraded, further compromising miRNA function and enhancing the expression of a large number of proteins required for cell cycle progression downstream of T-cell activation.
