Anjana Rao

Gene regulation mediated by calcium signals in T lymphocytes.

Modulation of many signaling pathways in antigen-stimulated T and B cells results in global changes in gene expression. Here we investigate the contribution of calcium signaling to gene expression in T cells using cell lines from two severe-combined immunodeficiency patients with several cytokine deficiencies and diminished activation of the transcription factor NFAT nuclear factor of activated T cells. These T cells show a strong defect in transmembrane calcium influx that is also apparent in their B cells and fibroblasts. DNA microarray analysis of calcium entry–deficient and control T cells shows that Ca2+ signals both activate and repress gene expression and are largely transduced through the phosphatase calcineurin. We demonstrate an elaborate network of signaling pathways downstream of the T cell receptor, explaining the complexity of changes in gene expression during T cell activation.

The Duration of Nuclear Residence of NFAT Determines the Pattern of Cytokine Expression in Human SCID T Cells

The expression of cytokine genes and other inducible genes is crucially dependent on the pattern and duration of signal transduction events that activate transcription factor binding to DNA. Two infant patients with SCID and a severe defect in T cell activation displayed an aberrant regulation of the transcription factor NFAT. Whereas the expression levels of the NFAT family members NFAT1, -2, and -4 were normal in the patients’ T cells, dephosphorylation and nuclear translocation of these NFAT proteins occurred very transiently and incompletely upon stimulation. Only after inhibition of nuclear export with leptomycin B were we able to demonstrate a modest degree of nuclear translocation in the patients’ T cells. This transient activation of NFAT was not sufficient to induce the expression of several cytokines, including IL-2, IL-3, IL-4, and IFN-γ, whereas mRNA levels for macrophage inflammatory protein-1α, GM-CSF, and IL-13 were only moderately reduced. By limiting the time of NFAT activation in normal control cells using the calcineurin inhibitor cyclosporin A, we were able to mimic the cytokine expression pattern in SCID T cells, suggesting that the expression of different cytokine genes is differentially regulated by the duration of NFAT residence in the nucleus.

Impaired NFAT regulation and its role in a severe combined immunodeficiency.

Severe Combined Immunodeficiency (SCID) is a primary immunodeficiency affecting T cells, B cells, or both. Whereas the clinical symptoms are uniformly dominated by recurrent infections, the molecular causes for SCID are very heterogeneous. Mutations in cell surface receptors, signal transduction molecules and transcription factors have been described, including the common gamma chain of the IL-2 (and IL-4, IL-7, IL-9 and IL-15) receptors, the kinase JAK-3, the epsilon and gamma chains of CD3, the protein tyrosine kinase ZAP-70, as well as CIITA and RFX5 involved in MHC class II gene expression. In this work we describe two infants with SCID whose T cells display a severe defect in T cell activation and cytokine transcription due to impaired activation of the transcription factor NFAT. We show that this defect in activation is not due to mutations in the NFAT proteins expressed in T cells or the phosphatase calcineurin which regulates the activation of NFAT. However, nuclear import of NFAT in response to T cell activation was severely compromised in the patients T cells. A modest degree of nuclear translocation of NFAT was achieved in the patients T cells when nuclear export was inhibited using lithium chloride. This low level of nuclear NFAT in the nucleus was not sufficient to compensate for the defect in cytokine production in the patients T cells. However, elevated levels of extracellular calcium led to an increase in cytokine gene transcription by the SCID T cells, suggesting that the underlying genetic defect in the patients involved calcium influx or the initiation of calcium signalling.

Peripheral Tolerance of T Cells in the Mouse

Publisher Summary Gene targeting in mice—inactivating single genomic loci at a time—has elucidated a predominant role for T cells in various autoimmune diseases. Mice carrying such mutations develop fatal systemic or organ-specific damage, the symptoms of which correlate well with many detrimental human autoimmune diseases. Genetic linkage analysis of human patients points to a complex multigenic basis for many autoimmune syndromes, and the associated genes or genetic regions show considerable overlap with genes linked to autoimmunity in mice. Although the knowledge of general “autoimmune factors” is limited at present, some of the genes identified could have diagnostic and therapeutic potential. Experimental access to cells and tissues in mice has allowed investigators to study perturbations in processes such as thymic selection, T cell homeostasis, differentiation, or activation that are shown to result in autoimmunity, and critical molecules and pathways of signal transduction have been identified.

The NFAT Family

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.