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TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4+ T-helper cell population in the resting (=naïve) and activated (=effector) state, and 3) two commonly used CD4+ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4+ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²+-dependent proliferation of primary CD4+ T-cells indicating that TRPC3 may be involved in Ca²+ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²+ entry.

Stimulation of the T-cell receptor (TCR) activatesCa 2؉ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca 2؉ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca 2؉ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca 2؉ currents as well as TCR-dependent Ca 2؉ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca 2؉ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.

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Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca 2+ dependence of proliferation and apoptosis in primary human CD4 + T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca 2+ entry through Ca 2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca 2+ ] i ) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4 + T cells. We also show that proliferation was very Ca 2+ sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca 2+ ] i levels of 90-120 nM. We conclude that very small changes in [Ca 2+ ] i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8 1 T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8 1 T cells, laying the groundwork for further understanding their potential role in T-cell function.Key words: Exocytosis . Immunological synapse . SNARE proteins Supporting Information available online IntroductionCytotoxic T lymphocytes (CTLs) kill pathogen-infected and/or tumorigenic cells. To do so, they first establish contact with the target cells. The interface between CTLs and target cells is termed the immunological synapse (IS). Formation of the IS is initiated when CD3/T-cell receptor (TCR) complexes on the surface of the CTL recognize antigens that are presented by the MHC class I molecules on the surface of the target cell. This initiates a cascade of signaling events including the clustering of TCR complexes at the IS. Finally, lytic granules (LGs) reorient towards the IS and the cytotoxic components contained therein are being released into the cleft at the contact zone. This constitutes the granulemediated pathway of target cell killing, which is one of the most important pathways used by CTLs for killing target cells [1][2][3]. Dysfunction in any step of LG release, including reorientation to the IS, priming, docking or fusion at the IS, may lead to lifethreatening immune disorders, such as familial hemophagocytic lymphohistiocytosis (FHL) [4]. Several molecules, which are essential for LG trafficking or fusion, have been identified: syntaxin11 [5][6][7], Munc13-4 [8], Munc18-2 [9, 10] and Rab27a [11,12]. Interestingly, most of those proteins either belong to the SNARE (soluble NSF attachment receptors) family or execute their function via interaction with SNARE proteins.SNAREs belong to a family of membrane proteins that have been implicated as the conserved core protein machinery required for all intracellular membrane fusion events that mediate intracellular trafficking [13,14]. There are at present 38 known members of the mammalian SNARE family [15]. SNAREs were initially classified functionally as v-SNAREs and t-SNAREs SHORT COMMUNICATIONÃ ...

Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids(TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, we have examined the influence of the TRL motif on the subcellular distribution of TRPC4 in human embryonic kidney (HEK) 293 cells. We have also analyzed the consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family for the cell surface expression of TRPC4. Using immunofluorescence microscopy, we found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. Our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane.