The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by isocitrate dehydrogenase (IDH) mutations promotes gliomagenesis via DNA and histone methylation. Here, we identify an additional activity of R-2-HG: tumor cell-derived R-2-HG is taken up by T cells where it induces a perturbation of nuclear factor of activated T cells transcriptional activity and polyamine biosynthesis, resulting in suppression of T cell activity. IDH1-mutant gliomas display reduced T cell abundance and altered calcium signaling. Antitumor immunity to experimental syngeneic IDH1-mutant tumors induced by IDH1-specific vaccine or checkpoint inhibition is improved by inhibition of the neomorphic enzymatic function of mutant IDH1. These data attribute a novel, non-tumor cell-autonomous role to an oncometabolite in shaping the tumor immune microenvironment.

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.

The following resources related to this article are available online at http://stke.sciencemag.org. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca 2+ ) influx through Ca 2+ release-activated Ca 2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca 2+ imaging, patch-clamp recordings and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H 2 O 2 ) on ORAI channel activity and human T helper lymphocyte (T H cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H 2 O 2 . The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T H cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T H cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T H cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T H cells may tune cellular responses under oxidative stress. Article Toolshttp INTRODUCTION Intracellular Ca2+ is a second messenger involved in the regulation of a diverse range of functions (1). One of the major Ca 2+ entry pathways into nonexcitable cells, such as lymphocytes and epithelial cells, is through ubiquitously expressed Ca 2+ release-activated Ca 2+ (CRAC) channels that are localized in the plasma membrane. Ca 2+ influx through CRAC channels is activated when inositol 1,4,5-trisphosphate (IP 3 ) triggers Ca 2+ release from intracellular stores in the lumen of the endoplasmic reticulum (ER) (2-4).The concomitant decrease in ER luminal Ca 2+ triggers accumulation of the ER Ca 2+ sensor protein stromal interaction molecule (STIM1) into puncta close to the plasma membrane (5, 6). These clustered STIM1 proteins directly activate Ca 2+ influx through CRAC channels, which are encoded by the Orai gene family (7-11). ORAI proteins contain four transmembrane domains with both N-and C-terminal intracellular domains (12, 13) that contain putative N-terminal calmodulinbinding domains and C-terminal coiled-coil motifs. Orai family members are highly homologous, and Orai1 and 3 are widely expressed at the messenger RNA (mRNA) level, with Orai2 showing a somewhat more restricted expression pattern (14, 15). ORAI1 proteins contain the longest intracellular N termini with two proline-rich regions an...

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TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.Although the family of transient receptor potential (TRP) 3 ion channels has originally been defined by their homology to their seminal member, the Drosophila TRP channel (1, 2), their 28 mammalian homologs have evolved into different subfamilies with very distinct characteristics and a range of different functions. Many members share only a very distant homology to the original TRP channel. Although members of one subfamily generally have certain structural motifs in common, e.g. six ankyrin repeat elements for the TRPV (transient receptor potential vanilloid) family (3-5), a C-terminal coiled coil (CC) domain for the TRPM channels and the TRP box and TRP domain, which are loosely conserved in all mammalian TRPs, except TRPP (transient receptor potential polycystic kidney disease, polycystin) and TRPA (transient receptor potential ANKTM1), these elements do not necessarily confer a similar function. Binding of PIP2 to the TRP domain, for example, appears to inhibit TRPV1 channels (6) but stimulates TRPM5, -7, and -8 as well as TRPV5 (7-10). Certain sensing modalities, such as thermosensation, are also not restricted to one subfamily. Altering activity depending on the temperature is utilized by TRPV1-4 but also by TRPM8, TRPA1, and TRPM2 (for reviews see Refs. 11-13). Here, an interchangeable, specific region has been found within the intracellular C terminus that allows TRPV1 and TRPM8 to sense temperature (14).In analogy to voltage-gated potassium channels, it is thought that four subunits need to assemble to form a functional channel. Experimental evidence for tetramer formation exists for TRPV1, TRPV2, and TRPV5/6 (15-18).Because there is hardly any structural motif besides the amino acids adjacent to the pore within TM5 and TM6 and the TRP dom...