Ca²⁺ signaling is the major intracellular signaling system found in almost all cell types in the animal kingdom, but also extending to plant cells and fungi (1, 2). Ca²⁺ signaling requires Ca²⁺ gradients across organellar membranes and across the plasma membrane (PM). ATP-driven Ca²⁺ pumps, such as sarco-/endoplasmic reticular Ca²⁺ ATPase (SERCA) and plasma membrane Ca²⁺ ATPase (PMCA), build up such Ca²⁺ gradients allowing for rapid Ca²⁺ release from organellar Ca²⁺ stores and/or Ca²⁺ entry across the plasma membrane upon cell stimulation.

Global Ca²⁺ signaling in the form of an increase in the free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) throughout the cytosol, most often extending into the nucleus, was first described in the 1980s (3). Such experiments were first performed in cell suspensions analyzed in a fluorimeter; however, with the advent of microscopic imaging technology, global Ca²⁺ signaling was also observed in single cells (4). With the increasing spatio-temporal resolution of microscopes and cameras, analyses of the dynamics of local Ca²⁺ signaling has become possible. In particular, the advent of confocal microscopy and the development of super-resolution imaging methods were major steps to allow for very high resolution in both space and time. Further, novel deconvolution approaches allow to increase signal-to-noise ratio even in dim raw images, adding another important step to disentangle fast local Ca²⁺ signaling events (5).

Recently, we reviewed functions and mechanisms of formation of Ca²⁺ microdomains (6). In summary, Ca²⁺ microdomains in which the rise in concentration reaches a few hundred nanomolar and extending on 1-3 microns were found in different cell types. Pioneering work by Peter Lipp, Martin Bootman and colleagues revealed elementary Ca²⁺ signal events, likely representing Ca²⁺ release from clusters of either ryanodine receptors (RYR), termed ‘sparks’ or d-myo-inositol 1,4,5-trisphosphate receptors (IP₃R), termed ‘puffs’. Smaller fundamental Ca²⁺ signal events that relate to Ca²⁺ release from single RYR, termed ‘quarks’ or IP₃R, termed ‘blips’ were also described (7–10). In addition to these local Ca²⁺ release events, Ca²⁺ sparklets were defined as local signals resulting from brief openings of plasma membrane Ca²⁺ channels (11–13).

More recently, Ca²⁺ microdomains occurring in confined spaces between closely apposed membranes have been described. Locally elevated Ca²⁺ concentrations arise around mitochondria-associated ER membranes (MAM) that provide privileged platforms for Ca²⁺ transfer from the ER into mitochondria (14). Similarly, sharp and short-lived Ca²⁺ increases can sometimes be observed at the ER-PM contact sites or junctions. These signals are dependent on Ca²⁺ entry from the extracellular medium through store-operated Ca²⁺ entry (SOCE). The latter mechanism relies on the stromal interaction molecules (STIM1 and/or STIM2) that are ER Ca²⁺ sensors regulating the activity of the Orai1 PM Ca²⁺ channels. Thus, upon local depletion of the ER Ca²⁺ store, Ca²⁺ dissociates from STIM, which allows recruitment of Orai1 to the junction and their gating (15–18). These channels are also referred to as “Ca²⁺ release activated channels (CRAC)”. The ER-PM junctions are areas in the cytosol where the ER gets really close to the PM, with a depth of approximately 15 nm and an extension of ~200 nm (18).

As proposed by 19, Ca²⁺ microdomains can be defined as “subcellular, rapid and localized high Ca²⁺ concentration regions that develop near open Ca²⁺ channels at the plasma membrane or internal stores”. Key factors that determine their specificity in triggering different signaling pathways in different cell types are their amplitude, frequency and duration. Variability in these factors leads to specific forms of global Ca²⁺ increases. The detailed characterization of Ca²⁺ microdomains is limited by the spatio-temporal resolution of available imaging techniques. Computational modelling has therefore much been used to further investigate their molecular origin. These studies rely on the theoretical tools that have been developed to describe global Ca²⁺ signaling (20), although they face specific problems related to the confined geometry and the steep gradients that need to be simulated (18). Moreover, stochastic approaches are sometimes required to take into account the local fluctuations in concentrations due to the small numbers of Ca²⁺ channels present in the microdomains (21–25).

Local Ca²⁺ entry via SOCE activates different cellular responses in different cell types depending on their spatio-temporal profiles. These include transcription factor stimulation in B cells by high amplitude Ca²⁺ spikes, NFAT activation in T cells by a sustained low amplitude signal and exocytosis stimulation in non-excitable cells by spontaneous repetitive short Ca²⁺ signals. It is known that the modulation of such localized Ca²⁺ signals involves clusters of ER (IP₃R or RYR) or PM (Orai1) Ca²⁺ channels, but the detailed interplay between these channels, including their molecular effectors, remain to be fully characterized.

Although most of the Ca²⁺ toolbox molecules, e.g. second messenger forming and degrading enzymes, Ca²⁺ mobilizing second messengers and their receptors, Ca²⁺ channels, Ca²⁺ storing organelles, and Ca²⁺ pumps are often ubiquitously expressed, the specific configuration of a particular cell type determines the exact mechanism of both local and global Ca²⁺ signaling. Thus, in this review, we will concentrate on a particular cell type that has been extensively characterized regarding mechanisms involved in the formation of Ca²⁺ microdomains, mammalian T-lymphocytes. The main molecules involved are listed in Table 1, for ease of reading. Over the past 10 years, Ca²⁺ microdomains at the ER-PM junctions have been extensively studied in these cells. Despite disadvantages of this cell model, e.g. small diameter of about 6-7 μm and a large nucleus as compared to the cytosol, as well as spherical shape with many stacked z-layers that potentially result in image blurring, development of a high-resolution Ca²⁺ imaging method combined with image refining by off-line deconvolution resulted in spatio-temporal resolution of 368 nm and 25 ms (26). Further, activation of the T cell receptor (TCR)/CD3 complex and co-stimulation of CD28 by small beads coated with monoclonal antibodies against CD3 and CD28 allowed for point-shaped activation of few TCR/CD3 complexes, thereby mimicking activation during formation of an immune synapse (26).

So far, two different signaling mechanisms involved in formation of Ca²⁺ microdomains in T cells were identified: (i) adhesion-dependent Ca²⁺ microdomains (ADCM) evoked by integrin-mediated IP₃ signaling and subsequent SOCE (Figure 1A), or (ii) Ca²⁺ microdomains evoked by TCR/CD3/CD28 stimulation (TDCM), proceeding via nicotinic acid adenine dinucleotide phosphate (NAADP) formation by dual NADPH oxidase 2 (DUOX2), and activation of RYR1 via the NAADP receptor hematological and neurological expressed 1-like protein (HN1L)/jupiter microtubule associated homolog 2 (JPT2). Hereafter, ADCM and TDCM will refer to the spatially restricted and short-lived Ca²⁺ increases occurring in the so-called microdomains. TDMC are amplified by SOCE (27) (Figure 1B) and purinergic signaling via P2X4 and P2X7 (28).

Long before the observation of ADCM, Weissmann and colleagues described global Ca²⁺ responses in Jurkat T cells that were triggered by integrin-mediated adhesion to extracellular matrix (ECM) proteins (29). These Ca²⁺ signals were dependent on the phosphorylation of focal adhesion kinase (FAK). FAK is thought to be a key component of the integrin-mediated signaling cascade (30) and evokes Ca²⁺ signals selectively at membrane rafts during the cell–matrix adhesion process (31). Stimulation of FAK subsequently activates phospholipase C-γ (PLC-γ) (32), which in turn generates IP₃ leading to a Ca²⁺ release from the ER by targeting IP₃ receptors (33). Local decrease in ER Ca²⁺ concentration then activates SOCE. Indeed, locally restricted Ca²⁺ microdomains in the absence of TCR stimulation were shown to be dependent on the expression of Orai1 and STIM1/2 (27). Interestingly, the NAADP/RYR1 axis was not involved in this process in primary T cells (27). However, another Ca²⁺ mobilizing second messenger, termed cyclic ADP-ribose (cADPR), which evokes Ca²⁺ signals through ryanodine receptor type 3 (34), was involved in global Ca²⁺ responses mediated by integrins in Jurkat T cells (35). Purinergic signaling via P2X4, is also involved in the formation of TCR independent Ca²⁺ microdomains in primary T cells (28). However, whether integrin mediated Ca²⁺ signaling evokes ATP release followed by purinergic signaling via P2X4 in an autocrine manner is not yet clear. Concerning the physiological role of adhesion-dependent Ca²⁺ microdomains, it has been suggested that they may facilitate full TCR-mediated activation (36) given that T cells form adhesive interactions with endothelial cells or ECM proteins during their migration from blood vessels into inflamed tissues (37).

TDCM require functional NAADP signaling in T cells (Figure 1B). A crucial role of NAADP in T cells was discovered by showing global and local Ca²⁺ signaling upon NAADP microinjection (38, 39; Dammermann & Guse; 2005; 26, 40). RYR was identified as a major Ca²⁺ channel in NAADP signaling in T cells (39; 41; 26, 27, 40). In addition, NAADP evoked Ca²⁺ entry secondary to Ca²⁺ release was also shown for the first time in T cells (39). The mechanism of NAADP evoked release of Ca²⁺ from intracellular Ca²⁺ stores started to being disentangled by the discovery of small cytosolic NAADP receptor/binding proteins, firstly as 22/23 kDa band labeled by a NAADP-photoaffinity probe (42) and finally by identifying the NAADP receptor/binding protein as HN1L/JPT2 in both T cells (43) and erythrocytes (44). Of note, Lsm12 was identified as another NAADP binding protein (45). HN1L/JPT2 co-localized with RYR in T cells as shown by STED super-resolution microscopy and co-immunoprecipitation (43), suggesting that NAADP bound to HN1L/JPT2 directly activates RYR, as hypothesized in the unifying hypothesis for NAADP action (46). The latter also includes, as further potential link between NAADP and activation of ion channels, the possibility of activation of two-pore channels (46). In addition to the Ca²⁺ releasing mechanism of NAADP, formation and degradation of NAADP are rather important for this signaling pathway. Along these lines, it was reported for T cells that NAADP formation within seconds upon TCR/CD3 stimulation (47) proceeds by oxidation of NAADPH to NAADP, catalyzed by DUOX2 (48). Rapid reduction of NAADP to NAADPH may be catalyzed in intact cells by glucose 6-phosphate dehydrogenase, that, together with DUOX2, constitutes a redox cycle for fast formation of NAADP and its ‘storage’ as inactive reduced NAADPH (48). Full degradation of NAADP is catalyzed by CD38 (to 2-phospho-ADPR) or by alkaline phosphatase (to NAAD) (49, 50). Crucial importance of NAADP signaling for Ca²⁺ microdomain formation in T cells was demonstrated by gene deletion or silencing of Duox2, Hn1l/Jpt2, or Ryr1 (26, 27, 43, 48).

Gene deletion of the major proteins of SOCE in T cells, STIM1 and 2, and Orai1, revealed that in addition to NAADP evoked release of Ca²⁺, also SOCE is essentially required for the formation of the local Ca²⁺ microdomains (27). Very recently, another amplification system for Ca²⁺ increases inside microdomains in T cells was discovered as autocrine ATP evoked activation of purinergic Ca²⁺ channels P2X4 and P2X7 (28).

TDCM are observed as soon as a few hundreds of millisecond post TCR/CD3/CD28 stimulation and last for approx. 15 to 25s (Figure 1B). Among the other initial signaling pathway active in T cells in this very early period of time, tyrosine phosphorylation of target proteins is certainly outstanding, e.g. by the tyrosine kinases ZAP70 and/or p56^lck (51). Experimental evidence for their involvement in TDCM was obtained: NAADP formation evoked by TCR/CD3 stimulation was completely blocked by tyrosine kinase inhibitor genestein (47). In contrast, during the first 15s, levels of IP₃ and diacylglycerol, both products formed from phosphatidylinositol 4,5-bisphosphate by phospholipase C-γ (52) are still quite low (53, 54) and do not significantly impact on Ca²⁺ microdomains, as demonstrated, at least for IP₃ evoked Ca²⁺ release, by IP₃ antagonism (26). Later on, between approx. 20 and 30s post TCR/CD3/CD28 stimulation, TDCM merge into global Ca²⁺ signaling. Major mechanisms involved in global Ca²⁺ signaling are (i) IP₃ evoked Ca²⁺ release between approx. 30s and 10 to 15 min (53), (ii) cyclic ADP-ribose mediated Ca²⁺ release started between 5 and 10 min and lasting for at least 60 min (55). SOCE via STIM1 and Orai1 is the major Ca²⁺ entry mechanism secondary to ER Ca²⁺ store depletion by IP₃ and cyclic ADP-ribose [reviewed in (56).

As detailed above, current techniques in microscopy allow to analyze both global Ca²⁺ dynamics and localized Ca²⁺ events. These observations however raise numerous questions about the molecular mechanisms involved in the foundation of Ca²⁺ microdomains. Some of these questions are best addressed by computational modelling, which is made possible thanks to an impressive quantitative characterization of the properties of ER-PM Ca²⁺ microdomains. In particular, models allow to predict the number and spatial arrangement of the channels inside the junction, which remains inaccessible with current experimental techniques.

A few studies have focused on modelling Ca²⁺ microdomains due to SOCE. In a series of pioneering studies (57–59), Ca²⁺ changes at the immunological synapse have been modelled in 1D and later in 3D. These early models focused on the impact of mitochondria on the Ca²⁺ influx through Orai, but most of them did not specifically address the detailed conditions related to the small-scale microdomains in the ER-PM junctions. To assess the impact of mitochondria relocalization to the immunological synapse on the spatial distribution of Ca²⁺ inside the cell, (60) developed a whole cell model considering specific geometries for mitochondria. The model predicted that the experimentally observed locations of this Ca²⁺-exchanging organelle, moving close to the PM, can account for the increase in Ca²⁺ around a 100 nm radius cluster of ORAI channels forming a Ca²⁺ microdomain. In the simulations, such local Ca²⁺ increases occur in conditions of massive ER Ca²⁺ depletion that correspond to situations encountered a few minutes after T cell stimulation, when mitochondria start to move along the cytoskeleton. In this study, the channel locations in the PM, ER or mitochondrial membranes are not specified and Ca²⁺ fluxes are homogeneously distributed across these membranes. The detailed impact of Ca²⁺ channel locations on the spatio-temporal profiles of Ca²⁺ changes inside the ER-PM junctions were analyzed in 1D by (18) and modelled in 3D by (61, 62) and (63, 64). In this section, we describe these models and their main relying assumptions, while their physiological outcomes are presented in the next section.

Model building can be divided in three steps. First, the biologically relevant variables that the model aims at simulating must be selected. Second, the spatial geometry in which these variables are evolving have to be defined, including the locations of organelles, channels, pumps, transporters, etc. Finally, well-chosen rate equations establish how the selected variables evolve in time in the elected sub-cellular configuration. As compared to global cell model, simulations of microdomains call for specificities especially when located around the mouth of a channel (20).

Concerning the choice of the variables, the formation of Ca²⁺ microdomains involve Ca²⁺ movements in the junctions, in the ER portion just beneath the junction (triggering SOCE) and in the extracellular medium. Given that the latter can be seen as an infinite source of Ca²⁺, its concentration can be considered as constant. Thus, Ca²⁺ concentrations in the junction and in the sub-PM ER compartment represent key variables. Because they dynamically regulate the junctional Ca²⁺ concentration, buffers should in principle be taken into account. Most models however ignore the effect of buffers because of the short time and length scales involved in microdomain formation. Because of the high Ca²⁺ concentrations and the small volume of the junction, local Ca²⁺ buffers are expected to be saturated (62, 65, 66). Similarly, Ca²⁺ diffusion is assumed to occur in an unbuffered cytosol (D_c = 220 μm²s^-1). When considering the relation between cell adhesion/stimulation and Ca²⁺ signaling, IP₃ and NAADP should also be involved because they modify ER and junctional Ca²⁺ by affecting the opening states of the IP₃Rs and RYRs, respectively. Although NAADP mobilizes lysosomal Ca²⁺ (67), the involvement of this organelle in the formation of T cells Ca²⁺ microdomains remains to be established (68). Instead of considering [IP₃] and [NAADP] explicitly, their effects can be modelled by changing the opening states of their receptors directly (63, 64). This simplification is however not valid when considering the transition from local to global Ca²⁺ signaling which involves considering diffusion and metabolism of the messengers. These variables and their interplays are schematically shown in Figure 1.

To predict the exact spatio-temporal dynamics underlying Ca²⁺ increases in microdomains, Ca²⁺ fluxes and diffusion must be simulated in a realistic computational description of the ER-PM junction configuration. A typical configuration, used with slight variations in (61, 62) and 63); 64), is schematized in Figure 2A. ER-PM junctions are regions of the cell where both membranes are closely apposed and correspond to the locations where STIM and Orai co-localize upon store depletion. In resting T cells, STIM2 pre-formed complexes with Orai localize at the ER-PM junctions (27). The distance between the two membranes has been estimated to be 10-20 nm in depth and ~200 nm in length (15, 18, 69, 70). This flat cylinder defines the junctional space, surrounded by the cytoplasmic space where Ca²⁺ concentration is assumed to remain constant given the limited amount of Ca²⁺ ions involved in the formation of microdomain. Similarly, the luminal space is arbitrarily divided in two regions: one close to the membrane facing the ER in which [Ca²⁺] changes dictate SOCE dynamics and one further away where [Ca²⁺] is assumed to remain constant due to fast replenishment from the rest of the ER.

Five Orai channels are placed in the PM, as estimated for Jurkat T cells (18). They are arranged on a ring with an inter-channel distance of 47 nm (61). It should be noticed that the number of Orai channels in the junction is cell-dependent, as one channel per junction is reported for HeLa cells (71). The number of SERCA pumps present in the junction remains unknown and was arbitrarily set to 10 in in (61, 62 and (63, 64). In the whole cell model of (60), 10⁵ SERCA’s spread over the entire surface area of the ER, which would also correspond to ~10 pumps per ER-PM junction assuming a homogenous distribution. However, model outcomes are practically independent of the numbers of SERCA’s that are found to exert little influence on the spatio-temporal dynamics of microdomain formation. In line with this finding, SERCAs were not included in the first studies of Ca²⁺ changes at the immunological synapse (57–59). IP₃Rs and RYRs with a well-defined spatial distribution were explicitly considered by Gil et al. (63, 64), because of the focus of these studies on SOCE possibly induced by moderate, local increases of IP₃ and NAADP. The spatial arrangement of the IP₃Rs (see Figure 3A) mimics the observations of (72) in HeLa cells, where clusters of IP₃Rs reside alongside the junction and facing the PM. In the absence of specific information, the same arrangement was also considered for RYRs. Indeed, when RYRs are placed inside the junction, which would correspond to the situation encountered in the cardiac dyadic clefts (73), simulated Ca²⁺ microdomains do not correspond to observations [see next section, (64).

How Ca²⁺ ions evolve in space and time in the ER-PM junction is fixed by partial differential equations (PDE), or by a stochastic reaction-diffusion scheme such as, for example, the Gillespie’s algorithm (74). Given a basal [Ca²⁺] in the 10-100 nM range and a volume of ~10^-17 - 10^-15 liter, the number of Ca²⁺ ions in the ER-PM junction is between 0.06 and 60. Because the magnitude of the concentration fluctuations is inversely proportional to the number of ions, it is expected that stochastic fluctuations arising from molecular interactions and diffusion do not average out, thus calling for the requirement of stochastic simulations. All the studies mentioned here above however used PDE’s. This huge simplification is based on previous studies that have compared stochastic and deterministic modelling of Ca²⁺ dynamics in other confined cellular subspaces, such as a dendritic spine or a cardiac dyadic cleft. These works have indeed shown that for an appropriate range of physiological relevant parameters, stochastic Ca²⁺ signaling is well described using a deterministic model (75). This conclusion holds as long as the gating of the channels is not influenced by the ions that they transport, as it would be the case in the presence of Ca²⁺-induced Ca²⁺ release (76–78). Fast and mobile buffers also increase the magnitude of the fluctuations in the microdomain (79). When modeling Ca²⁺ microdomains occurring at the ER-PM junction on time periods of a few seconds following T cell stimulation, these conditions are met, and a deterministic description is appropriate. The resolution of the PDE’s remains challenging for several reasons, including the steep concentration gradients or the existence of widely different time scales. The numerous computational studies of Ca²⁺ “puffs” and “sparks” (21–24, 80, 81) have also paved the way for the modelling of ER-PM Ca²⁺ microdomains. However, for puffs and sparks, there is no barrier for the diffusion of Ca²⁺ flowing through the ER Ca²⁺ channels. In contrast, Ca²⁺ microdomains in dyadic clefts share with ER-PM junctions the characteristic of being embedded between two closely apposed membranes and many conceptual problems related to the deterministic Ca²⁺ dynamics in such restricted spaces have been formally addressed in this case (75, 82). It is also possible to resort to compartmental model. Wieder et al. (78) have summarized the evolution of numerical simulation techniques developed until recently, which accurately approach the different spatial and temporal scales found in the analysis of Ca²⁺ microdomain formation. For T cell Ca²⁺ microdomains, McIvor et al. (62) resorted to Green functions while Gil et al. (63, 64) took profit of the high accuracy of the COMSOL Multiphysics software (83) [COMSOL Multiphysics^®]. Thanks to its powerful mesh dissection, solution methods and abundant post process operation, this software offers a trustable and relatively easily tractable way of modelling multiscale cellular processes.

Together with the increasingly accurate observations of Ca²⁺ dynamics in the ER-PM junctions, modelling studies have allowed to dissect Ca²⁺ signaling in these localized domains, and in particular the nature, number and spatial arrangement of the Ca²⁺ transporting channels that are at play at various stages following T cell stimulation. Samanta et al. [2015] were the first to provide a quantitative analysis of the Ca²⁺ profiles upon SOCE activation in a realistic 3D ER-PM junction. They showed that Orai clustering enables the Ca²⁺ sensors located on the ER surface to be exposed to Ca²⁺ levels considerably higher in the junction than in the nearby cytoplasm. In the same line, (62) focused on the Ca²⁺ spatial signatures in the ER-PM junction created by two different spatial arrangements of Orai channels and their impact in the ER refilling process: firstly, with an inter-channel distance of 39 nm (clustered) and secondly, of 65 nm (non-clustered), as shown in Figure 2B. They concluded that the spatial arrangement of the Orai channels significantly impacts Ca²⁺ changes in the junction. When clustered, junctional Ca²⁺ concentration rises from an initial value of 100 nM to 7 μM in the center of the junction, while this increase is limited to 4 μM in the non-clustered configuration (Figure 2C). In contrast, these changes do not impact on the rates of ER refilling because of the low K_D of SERCA pumps (~300 nM). Orai channels are assumed to remain open with a constant current of 2.1 fA (18) across each channel. At the mouth of the Orai channel Ca²⁺ concentrations around 60 μM are predicted (Figure 2D). The Ca²⁺ junctional concentration reaches its steady state already after 0.25ms, which highlights the temporal reactivity created by this spatial arrangement of channels and membranes.

Ca²⁺ microdomains in T cells appear under conditions of a full ER opposite to the conditions explored by (62). Yet they appear to be opposed to the PM suggesting that they originate directly at ER-PM junctions. Thus, their computational investigation has been performed in the same geometry as that proposed in (62) and schematized in Figure 2A. Diercks et al. (27) quantitatively characterized the two different types of Ca²⁺ increases in microdomains that precede global Ca²⁺ signaling in T cells. The first ones, termed ADCM, evoked by an interplay between IP₃R and/or SOCE and triggered by adhesion, are characterized by a duration of 44 ± 4 ms and a junctional average amplitude of 290 ± 12 nM. The second ones, termed TDCM, relying on RYR and SOCE and triggered by early TCR/CD3 stimulation, are much more frequent and have a duration of 64 ± 3 ms and a junctional average amplitude of 325 ± 11 nM [see 6, for review].

Computational modeling is increasingly used in cell biology to optimize the information that can be gained from the ever-improving techniques in imaging microscopy. In the field of ER-PM Ca²⁺ microdomains, this synergy has allowed to predict the number and spatial arrangement of Orai channels in the junction (18, 61), and to highlight the relatively minor role played by ER refilling by SERCA pumps in controlling the amplitude of the Ca²⁺ increase and the spatial spread of Ca²⁺ microdomains (62). When considering the ER Ca²⁺ channels surrounding the junction, a comparison between simulation results and high-resolution Ca²⁺ imaging uncovered the main role played by ER local depletion induced by weak IP₃ or NAADP signaling in triggering the formation of adhesion-dependent and TCR/CD3-dependent Ca²⁺ microdomains in T cells (63, 64). In the future, it will be necessary to resort to observations-based multiscale modeling to dissect the relationship between local and global Ca²⁺ signaling, which could emphasize the key role played by the PMCA’s and the mitochondria in the T cell Ca²⁺ dynamics beyond the first seconds after stimulation (60). Despite the corresponding computational challenge, this effort is necessary to reveal how the versatile Ca²⁺ signaling pathway fine-tunes T cell immune responses.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.