Ariel Quintana

T cell activation requires mitochondrial translocation to the immunological synapse

T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values <200 nm. Consequently, mitochondria close to the IS took up more Ca2+ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca2+ influx across the plasma membrane and Ca2+-dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.

Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation

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.

Sustained activity of calcium release-activated calcium channels requires translocation of mitochondria to the plasma membrane.

A rise of the intracellular Ca2+ concentration has multiple signaling functions. Sustained Ca2+ influx across plasma membrane through calcium release-activated calcium (CRAC) channels is required for T-cell development in the thymus, gene transcription, and proliferation and differentiation of naïve T-cells into armed effectors cells. Intracellular Ca2+ signals are shaped by mitochondria, which function as a highly dynamic Ca2+ buffer. However, the precise role of mitochondria for Ca2+-dependent T-cell activation is unknown. Here we have shown that mitochondria are translocated to the plasma membrane as a consequence of Ca2+ influx and that this directed movement is essential to sustain Ca2+ influx through CRAC channels. The decreased distance between mitochondria and the plasma membrane enabled mitochondria to take up large amounts of inflowing Ca2+ at the plasma membrane, thereby preventing Ca2+-dependent inactivation of CRAC channels and sustaining Ca2+ signals. Inhibition of kinesin-dependent mitochondrial movement along microtubules abolished mitochondrial translocation and reduced sustained Ca2+ signals. Our results show how a directed movement of mitochondria is used to control important cellular functions such as Ca2+-dependent T-cell activation.

Calcium-dependent activation of T-lymphocytes

Activation of T-lymphocytes requires stimulation of T-cell receptors (TCR) and co-stimulatory signals. Among different signalling cascades, TCR engagement induces Ca2+ entry through plasma membrane Ca2+ channels, which is an indispensable step for T-cells to expand clonally and to acquire effector functions. The Ca2+ channels are activated by depletion of Ca2+ stores and are called Ca2+ release-activated Ca2+ (CRAC) channels. Ca2+ influx through CRAC channels is also controlled, directly or indirectly, by K+ channels, Ca2+-ATPases, mitochondria, endoplasmic reticulum and Ca2+ buffers. We review the functional implications of these transporters, organelles and buffers and develop a model of Ca2+ signal generation that depends mainly on their relative mutual localization. This model offers the possibility of controlling amplitude and kinetics of Ca2+ signals in T-cells. Decoding of various Ca2+ signals allows differential activation of the transcription factor families nuclear factor of activated T-cells (NFAT), nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Variation of amplitude and kinetics of Ca2+ signals thus is an important mechanism for modulating the specificity of T-cell responses.

Mitochondria Positioning Controls Local Calcium Influx in T Cells

Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.

Calcium dependence of T cell proliferation following focal stimulation

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 Ca2+ 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 Ca2+ entry through Ca2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca2+]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 Ca2+ sensitive in the range 90–120 nM, whereas apoptosis was basically constant for [Ca2+]i levels of 90–120 nM. We conclude that very small changes in [Ca2+]i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

The immunological synapse controls local and global calcium signals in T lymphocytes

Summary:  Cell polarization is a key feature of T‐cell function. The immunological synapse (IS) between T cells and antigen‐presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re‐localizations have already been linked with certain T‐cell functions. One key step for T‐cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T‐cell polarization for calcium signals and calcium‐dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS‐dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals leads to a model how the IS controls local and global calcium signals and calcium‐dependent T‐cell functions. We develop a theoretical formalism based on existing spatiotemporal calcium dynamic simulations to better understand the model in the future and allow further predictions which can be tested by fast, high resolution live‐cell microscopy.

TRP Channels in Lymphocytes

TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.

Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.

Nonsteroidal Anti-inflammatory Drugs Inhibit Vascular Smooth Muscle Cell Proliferation by Enabling the Ca2+-dependent Inactivation of Calcium Release-activated Calcium/Orai Channels Normally Prevented by Mitochondria

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+.