Faculty Bibliography

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells

The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.

BACKGROUND: Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott-Aldrich syndrome (WAS). The transcription factors nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. OBJECTIVE: To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). METHODS: T cells from WASP-/- mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kappaB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kappaB-dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. RESULTS: Nuclear translocation and activity of NF-kappaB were normal in T cells from WASP-/- mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. CONCLUSION: These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS.

Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.

Engagement of the TCR triggers sustained Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1-2% of the normal level of Ca(2+) influx and severely impaired T cell activation. The lack of Ca(2+) influx is not due to deficient regulation of Ca(2+) stores or expression of several genes implicated in controlling Ca(2+) entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca(2+) entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells

Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor-induced interleukin (IL)-4 secretion by SLAM(-/-) CD4(+) cells is down-regulated, whereas interferon gamma production by CD4(+) T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor that regulates signals transduced by the major LPS receptor Toll-like receptor 4 on the surface of mouse macrophages. A defective macrophage function resulted in an inability of SLAM(-/-) C57Bl/6 mice to remove the parasite Leishmania major. We conclude that the coreceptor SLAM plays a central role at the interface of acquired and innate immune responses.