Calcium signal dynamics in T lymphocytes: Comparing in vivo and in vitro measurements

Seminars in Cell and Developmental Biology 94 (2019) 84–93

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Calcium signal dynamics in T lymphocytes: Comparing in vivo and in vitro measurements Kim S. Friedmann1, Monika Bozem1, Markus Hoth

T

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Department of Biophysics, Center for Integrative Physiology and Molecular Medicine, Medical Faculty, Saarland University, Homburg, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Calcium Immunological synapse Murine versus human lymphocytes Lymph node In vivo versus in vitro Genetically encoded Ca2+ indicators

Amplitude and kinetics of intracellular Ca2+ signals ([Ca2+]int) determine many immune cell functions. To mimic in vivo changes of [Ca2+]int in human immune cells, two approaches may be best suited: 1) Analyze primary human immune cells taken from blood under conditions resembling best physiological or pathophysiological conditions. 2.) Analyze the immune system in vivo or ex vivo in explanted tissue from small vertebrate animals, such as mice. With the help of genetically encoded Ca2+ indicators and intravital microscopy, [Ca2+]int have been investigated in murine T lymphocytes (T cells) in vivo during the last five years and in explanted lymph node (LN) during the last 10 years. There are several important reasons to compare [Ca2+]int measured in primary murine T lymphocytes in vivo and in vitro with [Ca2+]int measured in primary human T lymphocytes in vitro. First, how do human and murine data compare? Second, how do in vivo and in vitro data compare? Third, can in vitro data predict in vivo data? The last point is particularly important considering the many technical challenges that limit in vivo measurements and to reduce the number of animals sacrificed. This review summarizes and compares the results of the available publications on in vivo and in vitro [Ca2+]int measurements in T lymphocytes stimulated focally by antigen-presenting cells (APC) after forming an immunological synapse.

1. Introduction This review is focused on kinetic measurements of cytosolic Ca2+ concentration ([Ca2+]int) in T lymphocytes (also called T cells) and does not discuss molecular mechanisms generating Ca2+ signals. There are many excellent reviews on molecular mechanisms of T lymphocyte Ca2+ signaling [1–10] and we will therefore limit ourselves to the absolutely necessary facts on molecular mechanisms for Ca2+ homeostasis. In contrast, there are only few reviews on Ca2+ kinetics and even fewer on kinetics of [Ca2+]int in T lymphocytes comparing in vivo and in vitro data (see [11] for a detailed summary of intravital analysis of T lymphocyte activation). Considering the relevance of Ca2+ signaling for immune cell function, we believe that quantification of [Ca2+]int kinetics, in particular in in vivo models, is of utmost

importance. It is of course impossible to analyze the immune system in humans on a cellular level in vivo. But how can we mimic physiological and pathophysiological immune functions in humans? In our opinion, there are two main approaches. 1) Study the immune system in vivo or in explanted tissue ex vivo in small vertebrates like mice. 2) Study primary human immune cells taken from blood under conditions resembling best physiological or pathophysiological conditions. It was always an important goal to analyze in particular [Ca2+]int changes and signals in vivo in mice or in explanted lymph nodes (LN) from mice. For many reasons this is a technical challenge. Two major breakthroughs greatly facilitated in vivo [Ca2+]int measurements. First, three papers published in Science in 2002 reported about live cell imaging technologies to analyze dynamic cellular interactions in

Abbreviations: Ag, antigen; APC, antigen-presenting cell; [Ca2+]int, intracellular Ca2+ concentration; CTL, cytotoxic T lymphocyte; Cln, calcineurin; CRAC channel, Ca2+ release-activated Ca2+ channel; DAG, diacylglycerol; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; ER, endoplasmic reticulum; GC, germinal center; IP3R, inositol 1,4,5 tris-phosphate receptor; IS, immunological synapse; LN, lymph node; MCU, mitochondrial Ca2+ uniporter; MHC-I and II, major histocompatibility complex I and II; NCX, Na+/Ca2+ exchanger; NFAT, nuclear factor of activated T cell; PIP2, phosphatidylinositol-4,5-bisphosphate; PLCγ, phospholipase Cγ; PMCA, plasma membrane Ca2+ ATPase; SEA, Staphylococcus enterotoxin A; SERCA, sarco-/endoplasmic Ca2+-ATPase; STIM, stromal interaction molecule; TCR, T cell receptor; TFH, follicular helper T cells; Treg, regulatory T cells ⁎ Corresponding author at: Department of Biophysics, Center for Integrated Physiology and Molecular Medicine, Building 48, Saarland University, D-66421 Homburg, Germany. E-mail address: [email protected] (M. Hoth). 1 Contributed equally. https://doi.org/10.1016/j.semcdb.2019.01.004 Received 9 November 2018; Received in revised form 18 December 2018; Accepted 5 January 2019 Available online 11 January 2019 1084-9521/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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slightly migrate, whereas when matured, increase motility and thus increase probability to meet and activate T cells. DC (along with B cells and macrophages) function as antigen-presenting cells (APC), expressing antigen on major histocompatibility complexes (MHC). MHC-I complexes present antigens against intracellular pathogens, self-antigens (tolerance) and are recognized by CD8+ T cells. MHC-II complexes on the other hand bind antigens, which are derived from exogenous sources and induce immune responses in CD4+ T cells [36]. Maturation and proliferation of T cells is initiated by (multiple) contacts with APC. Certain specialized areas within LN and spleen are called germinal centers [37]. Here B cells undergo further proliferation and differentiation to mutate their antibody arsenal. Continuously proliferating cells which form different clones and express variable antibodies, are named centroblasts; after stopping proliferation, they are called centrocytes. As such they are specifically selected by follicular helper T cells (TFH) and follicular DC. Selection includes survival of B cells expressing high-affinity receptors and also apoptosis of centrocytes, which cannot bind antigen and therefore do not receive survival signals from TFH cells and follicular DC. This, “affinity maturation”-called process can adapt to the immunological requirements of the whole organism.

lymphatic tissue [12–14]. Many important publications in explanted LN or in vivo in the mouse were to follow, including two of special interest for the present review. They defined the sequential phases of T cell responses to antigen-presenting dendritic cells (DC) in vivo [15] or in explanted LN [16]. At least three consecutive steps of T cell-DC interactions were described, ranging from short, probably stochastic and transient encounters (phase I) to stable contacts with cytokine production by T cells, potentially involving T cell clusters (phase II) and finally to a proliferative stage with high migratory potential (phase III) [15,16]. Much of the progress in this fast-developing field is summarized by Germain and colleagues [17]. The second major breakthrough to allow long-term dynamic [Ca2+]int measurements was the development of genetically encoded Ca2+ indicators (GECI) based on GFP, which started already in 1997 by two different groups [18–20]. Today, these include among others calmodulin-based GCamP6 sensors [21,22] and troponin-based Twitch sensors [23], which partly allow ratiometric measurements and some of which have already been used to create transgenic reporter mice. Detailed summaries of GECI use for in vivo applications can be found in [11,22,24,25]. Researching the literature for [Ca2+]int data in in vivo models or in explanted tissue, we found by far most publications for murine T lymphocytes and only very few or none for other immune cells. Thus our review is exclusively focused on [Ca2+]int data in T lymphocytes with the goal to compare in vivo and ex vivo data from explanted LN with in vitro data available for primary human and murine T lymphocytes and T cell lines. To avoid confusion, we define the following terms: in vivo means in lymphatic tissue still connected to the living animal, explanted tissue (ex vivo) means measurements in lymphatic tissue not connected any more to the living animal, in vitro refers to either primary T lymphocytes from human, mice or to cell lines. Before summarizing the [Ca2+]int data, we need to shortly introduce basics about lymph nodes, the main organ to measure [Ca2+]int in T cells. Furthermore, we need to briefly introduce Ca2+ channels, transporters and other mechanisms which control [Ca2+]int in T lymphocytes.

1.2. The immunological synapse and Ca2+ transport in T lymphocytes The immunological synapse (IS) is the central communication and signaling unit between T lymphocytes and APC [38], and it controls local and global Ca2+ signaling in T lymphocytes [5]. In almost all cases, [Ca2+]int measurements in vivo and ex vivo in explanted LN are performed in T lymphocytes stimulated focally through an IS by APC (Fig. 1A). Therefore we will focus the review mostly on [Ca2+]int data measured through focal stimulation. The vast majority of publications on non-focal T cell stimulation by Ca2+ releasing agents like thapsigargin, a combination of Ca2+ ionophores and phorbolesters, lectins or dissolved antibodies against the TCR are largely ignored in this review. Since it is sometimes not possible to work with clonal primary human or murine T cells, two other focal T cell stimulation methods are commonly employed instead in vitro in primary cells: an IS formed with APC by presenting a super antigen on MHC-II like the Staphylococcus enterotoxin A (SEA) antigen (Fig. 1B) or an artificial focal stimulation with antibody-coated beads (Fig. 1C). Ca2+ transporters, channels, pumps and mechanisms relevant for T lymphocyte physiology and pathophysiology have been summarized and evaluated in numerous reviews [1–10]. We will therefore only briefly recap the main Ca2+ transporters, channels and pumps relevant in T cells (Fig. 2). Originally described in mast cells, Ca2+ release-activated Ca2+ (CRAC) channels are the main Ca2+ entry route in T cells [39–41]. Cloning of their main components STIM [42,43] and Orai [44–46] facilitated the molecular analysis of Ca2+ entry in T cells and other cells and guided molecular mechanisms of CRAC/Orai pathologies (as summarized in [2]). Depletion of Ca2+ from endoplasmic reticulum (ER) leads to clustering of STIM and Orai and to activation of CRAC channel-dependent Ca2+ entry. In T cells, Ca2+ release from the ER occurs mainly through TCR dependent activation of IP3 receptors in the ER but other messengers and release channels may be involved (as summarized in [8]). The importance of Orai1 Ca2+ channels for T cell function is highlighted by Maul-Pavicic et al. [47] and Klemann in 2017 [48], who reported in ex vivo studies of primary patient material that a deficiency in Orai1 expression results in a severe impairment of CTL and NK cell-mediated cytotoxicity. Similar results were obtained with NK cells from STIM1 deficient patients [49]. In CD8+ T cells it is probably easier to directly link [Ca2+]int analysis with cytotoxicity of single cells than in CD4+ T cells, in which “helping” functions often require at least several hours if not days. As in most cell types, sarco-/endoplasmic Ca2+ ATPases refill the ER. The Ca2+ ATPase 4b is the main Ca2+ export mechanism across the plasma membrane [50]; Na+/Ca2+ exchangers apparently do not have any function in T cell lines [50,51] but their role has not been tested yet

1.1. Lymph nodes Lymph nodes (LN) are part of the fluid circulation systems of the body, and they are connected to both, the blood and the lymph system. Arteries and veins transport blood to and from each LN. The afferent and efferent vessels are the connections to the lymphatic system. LN, together with the spleen, belong to the secondary lymphoid organs; they are localized within vessels collecting lymphatic fluid which partly originates from blood capillary extravasation into the interstitial space and partly from metabolic products secreted by the cells of the parenchymal tissues. LN are well-organized organs which, among various other functions, are of preeminent importance for the immune defense. Homing of immune cells to and within LN is highly regulated by diverse parameters, including chemokines [26,27], plasma membrane structures [28], cytoskeletal structures [29,30], products of energy metabolism and multiple other factors [31,32]. Additionally, it has been shown that changes in [Ca2+] in- and outside of the cells are of superior importance for interrelated processes leading to differentiation and maturation of immune cells in the LN [33,34]. Immune cells, like DC, T lymphocytes, B cells, natural killer (NK) cells and monocytes/macrophages are residing in LN, where they interact, differentiate and mature to become professional effector cells of the adaptive immune system. As immature/naïve cells, produced in the thymus or bone marrow, they are transported to the LN via blood or lymphatic vessels [31]. Within the LN, T cells, NK cells and monocytes actively migrate and meet other cells of the same or another type. DC are more stationary, forming multiple dendrite-like protrusions, with which they directly contact other cells in the immediate neighborhood [16,35]. It was shown in murine LN in vivo that immature DC only 85

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Fig. 1. Scheme showing three different types of focal T cell stimulation through an immunological synapse. (A) Physiologically, T cells are stimulated by antigenpresenting cells (APC), for instance mature dendritic cells (DC) by an MHC-antigen-TCR complex involving co-receptor activation. (B) The focal aspect of this stimulation can be mimicked by a super-antigen, like the Staphylococcus enterotoxin A (SEA), which crosslinks MHC-II receptors with TCR of a T cell. (C) An artificial T cell receptor-ligand complex can be achieved by antibody (often against CD3 and CD28)coated beads. Ag, antigen; TCR, T cell receptor; MHC-I and II, major histocompatibility complex I and II.

in detail in primary T cells. Additionally, the mitochondrial uniporter MCU [52,53] transports Ca2+ into mitochondria, which do also express functional Na+/Ca2+ exchangers important for Ca2+ export from

mitochondria [54]. The latter ones can in principle also run in the reverse mode to facilitate Ca2+ entry into mitochondria [55]. Next to Orai channels, many other Ca2+ channels have been proposed in T cells

Fig. 2. Schematic overview of Ca2+ transporters, channels and other [Ca2+]int-regulating mechanisms in T cells stimulated through an antigen-presenting cell (APC). The focus is on store-operated Orai Ca2+ channels, their activation and on additional regulation of [Ca2+]int by other transporters. Also ion channels, like TRP, CaV, P2X receptors and Piezo channels might accomplish Ca2+ entry, but details on their respective contributions to T cell [Ca2+]int have not been included in the present review. Ag, antigen; Cln, calcineurin; DAG, diacylglycerol; IP3R, inositol 1,4,5 tris-phosphate receptor; MHC-I, major histocompatibility complex I; MCU, mitochondrial calcium uniporter; NFAT, nuclear factor of activated T cells; NCX, Na+/Ca2+ exchanger; PIP2, phosphatidylinositol-4,5-bisphosphate; PLCγ, phospholipase Cγ; PMCA, plasma membrane Ca2+ pump; SERCA, sarco-/endoplasmic reticulum Ca2+-ATPase, TCR, T cell receptor. 86

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to contribute to Ca2+ entry but ion current measurements and clear functions need to be defined in more detail (compare [8,56]). Ca2+ sensing receptors are involved in inflammation [57,58] but are probably not expressed in T lymphocytes.

colleagues observed spontaneous irregular baseline spiking in the absence of any stimulus. Under this condition, T cells moved fast with large displacements. Injection of an ovalbumin peptide antigen into the mouse induced T cell-DC contacts within minutes, loss of T cell migration and a rise in T cell [Ca2+]int. [Ca2+]int elevations in T cells were persistent for tens of minutes with occasional oscillations or transitions between different levels. In a second approach, mice deficient of the recombination-activating gene (Rag2−/−) were used which lack endogenous T and B cells [73] and develop a severe combined immune deficiency (SCID) phenotype. Twitch-1CD transduced MOG (myelin oligodendrocyte glycoprotein) peptide-specific T cell clones from C57BI/6 mice (called 2D2 T cells) [74] were transferred to Rag2−/− mice and Ca2+ responses were recorded during the peak of EAE. The experiments provide evidence for Ca2+ signals during onset EAE and peak EAE with some cells migrating and others being arrested. The authors referred to the Ca2+ signals as partly being oscillatory, sustained or “differential”. Considering the challenge to measure in vivo Ca2+ signals due to a very modest signal to noise ratio and limited time resolution, it is very difficult in particular in this EAE model approach to quantify the Ca2+ signals. Similarly to Mues et al. [71], Kyratsous et al. [75] also analyzed Ca2+ signals in encephalitogenic T cells in an EAE model. They combined a modified Twitch-1 Ca2+ sensor with fluorescent nuclear factor of activated T cells (NFAT). In vitro activated T cells were injected into rats and were first observed in secondary lymphatic tissue where only short lived Ca2+ transients (about 1 min long) were occasionally observed in the absence of autoantigen. After exiting lymphatic tissue and crossing the blood-brain barrier in the absence of any Ca2+ signaling, T cells interacted with APC in the leptomeningeal space. Contacts with many but not all APC were paralleled by sustained Ca2+ signals and NFAT translocation indicating substantial T cell activation. [Ca2+]int showed a plateau-like behavior between 3 and 120 min with occasional Ca2+ transitions between the elevated state and baseline levels. In 2014, Shulman et al. [76] measured Ca2+ signals in germinal centers (GC) of popliteal LN in vivo by two-photon laser scanning microscopy. T follicular helper (TFH) cells from OT-II mice were adoptively transferred into congenic mice and primed with ovalbumin peptide antigen. To monitor Ca2+ signals in GC, TFH cells from OT-II DsRed+/GCaMP3+ mice were imaged in GC of popliteal LN during B cell selection. GCaMP3/DsRed ratios were higher in ovalbumin peptidetreated mice with most of the cells displaying sustained [Ca2+]int rises over tens of minutes. Ca2+ signals did not drop during the observation time. Ca2+ spikes were also occasionally observed in some cells but were not the predominant Ca2+ signal type. The authors report a small drop of migration velocity during Ca2+ signaling but overall, migration behavior of TFH cells was rather [Ca2+]int independent. In addition to the sustained Ca2+ signals in the presence of ovalbumin peptide antigen, TFH cells also displayed short Ca2+ transients correlated with B cell contact. In parallel to the Ca2+ signals, Ca2+ dependent cytokine production (IL-4, IL-21) was also observed in TFH cells.

2. [Ca2+] in T lymphocytes 2.1. Early fluorescence-based methods to measure [Ca2+] in T lymphocytes A potential role for [Ca2+]int in lymphocyte function was already proposed in 1964 [59] but it was not until 1982 that first [Ca2+]int measurements were carried out in mouse thymocytes, primary lymphocytes from pig LN and B lymphocytes by Tsien and colleagues [60–62]. The authors used the non-ratiometric dye Quin2 [62], which is excited at 339 nm and had to be applied at high concentrations to overcome cell auto-fluorescence when measuring lectin-induced [Ca2+]int rises in lymphocytes and thymocytes. Antigen-specific rapid Ca2+ signals measured with Quin2 and Indo-1 in human T-cell clones confirmed [Ca2+]int increases but did not show kinetics [63,64]. Quin2 was initially also used to monitor Ca2+ signals following antibody stimulation with OKT-3 against CD3/TCR in human Jurkat T cells [65,66], which is the most widely used T cell line to measure Ca2+ signals. The initial studies revealed sustained Ca2+ plateaus following TCR stimulation. While being used in many of the initial [Ca2+]int measurements in many cell types, Quin2 was quickly replaced by the ratiometric dyes Fura-2 (ratiometric excitation) and Indo-1 (ratiometric emission), which were also generated by the Tsien lab [67]. These dyes not only show “wavelength shifts upon Ca2+ binding”, which enables ratiometric Ca2+ imaging but are also characterized by much brighter fluorescence, higher affinity for Ca2+ and better selectivity against other divalent cations (in particular Mg2+) than Quin2 [67]. Fura-2 and Indo-1 are successfully used for Ca2+ imaging in primary immune cells up to date, probably because they are relatively easy to use and provide an extremely good dynamic range for [Ca2+]int measurements. Shortly after the first [Ca2+]int measurements in thymocytes and lymphocytes [60–62], Poenie and colleagues already described sustained [Ca2+]int signals in alloreactive specific murine cytotoxic T lymphocyte (CTL) clones stimulated by antigen-specific lymphoma cells [68]. CTL were loaded with Fura-2 and showed sustained [Ca2+]int elevations. There are, however, two significant disadvantages when using Indo1 and Fura-2: UV light is needed to excite the dyes, which is harmful to cells, and both dyes bleach/are transported out of the cytosol, Indo-1 more than Fura-2, in particular at higher temperatures (i.e. 37 °C). Modified variants of the dyes applied at higher wavelength (i.e. FuraRed) [69] or with reduced bleaching/transport out of the cell (FuraPE3) [70] exist, and are being used in lymphocytes. 2.2. [Ca2+] measurements in T lymphocytes in vivo In 2013, Mues et al. [71] used a genetically-encoded Ca2+ indicator (GECI) to measure Ca2+ signals in vivo in T cells in peripheral LN and in an area called leptomeningeal space, which is considered a main entry port for T cells into the central nervous system during experimental autoimmune encephalomyelitis (EAE). To quantify [Ca2+]int, the ratiometric troponin C-based CFP-cpCitrine FRET Ca2+ sensor TN-XXL [72] was optimized for expression in T cells by codon diversification und truncation of the retroviral vector size [71]. Despite its efficient expression in T cells in vivo, Mues and colleagues could only report “scarce and faint (Ca2+) signals”, which prompted them to switch to an advanced version of TN-XXL, termed Twitch-1 [23]. Twitch-1 is a FRET-based sensor with a Kd for Ca2+ of about 250 nM; it was also codon-diversified (CD) to optimize T cell expression (called Twitch1CD). In vivo [Ca2+]int measurements were performed in Twitch-1CDexpressing T cells in popliteal LN of anaesthetized OT-II mice. Mues and

2.3. [Ca2+] measurements in T lymphocytes ex vivo in explanted LN In 2005, Bhakta et al. developed a murine thymic slice preparation to compare [Ca2+]int signals in thymocytes during positive selection and under non-selecting conditions [77]. Thymi were prepared from C57BL/6 mice (non-selecting conditions) or from B10.BR/SgSn mice (positive selecting conditions) and indo-PE3-loaded thymocytes from 5C.C7 TCR transgenic mice were allowed to enter the slice. [Ca2+]int signals were monitored and quantified by ratiometric measurements with Two-photon microscopy. The authors observed high motility of thymocytes under non-selecting conditions with resting [Ca2+]int up to 200 nM and infrequent occasional Ca2+ spikes up to 500 nM. These Ca2+ spikes should not result from specific TCR interaction but may have been caused by adhesion mediated processes through integrins. Under positive selecting conditions, Bhakta et al. [77] observed low 87

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[Ca2+]int values in motile and high [Ca2+]int values in immotile thymocytes. High [Ca2+]int was characterized by irregular oscillations/ transitions between resting [Ca2+]int and 1000 nM. The first detailed measurements of [Ca2+]int in explanted LN was presented by Wei et al. [33]. They used Two-photon microscopy of Indo-1-labelled CD4+ T cells and compared Ca2+ signals during the early phase of T cells encountering antigen-presenting DC and under inflammatory conditions, evoked by cytokines and antigen-specific immune responses. When T cells were observed under non-stimulated conditions, in the absence of antigens, transient and infrequent Ca2+ signals occurred, which were paralleled by migration with reduced velocity and finally arrest. Velocity was further reduced upon addition of inflammatory stimuli or specific antigen, showing that T cell arrest might be triggered by TCR-dependent and TCR-independent factors. After having formed a stable contact with an APC, which could last for hours, T cell [Ca2+]int was elevated and occasional irregular spikes but not regular Ca2+ oscillations were superimposed on the elevated [Ca2+]int level. Elevated levels persisted for more than 30 min, also when the T cell left the DC and did not visibly contact another DC. The authors conclude that “sustained increases in basal [Ca2+]int and spiking frequency constitute a Ca2+ signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.” One important factor to control [Ca2+]int elevations is supposedly the antigen potency. This is not easily determined in vivo. Skokos et al. [78] tested low, medium and high antigen potency in vivo and in vitro on Ca2+ signals in T cells loaded either with Fura-2 or Fluo4 (the latter one in explanted murine LN). For this, the properties of DC peptideMHC complexes had been artificially modified, to generate TCR agonists ranging from “super-stimulatory” to non-stimulatory. The in vitro results of the paper show that only DC expressing high-potency antigen elicited significant Ca2+ responses in naïve T cells. Further in vitro experiments, using a transmigration assay, revealed that T cells stopped migration, after Ca2+ influx had been induced by Ca2+ store depletion from the ER, even when no antigen was present. To test if in vitro results could be confirmed in vivo, T cells, loaded with the fluorescent Ca2+ indicator Fluo4, were injected into a Vβ3Vα11 TCR-transgenic mouse. After T cells had reached the inguinal LN, they were observed in vivo. T cells showed an instantaneous elevation of [Ca2+]int, when high-potency antigens were presented. Low- and medium-potency antigens did not induce significant [Ca2+]int levels above control values. T cells did also not change migration speed. In summary, T cell migration speed and [Ca2+]int depended on each other and are determined by the potency of the presented antigen. In vitro and in vivo experiments produced similar results. The dependency of [Ca2+]int on antigen strength was confirmed by Waite et al. [79] in CD4+ T cells analyzed in explanted spleen. T cells were from DO11.10 mice, loaded with cell tracker orange CMRA to identify them and Fluo4 to qualitatively monitor [Ca2+]int. Soluble ovalbumin peptide concentrations (between 1 and 50 μg) elicited dosedependent [Ca2+]int elevations. In addition, the authors employed naïve or CD4+ effector T cells from mice in which CRAC-operated Ca2+ influx (SOCE) was blocked, either because they were lacking STIM proteins or they expressed a dominant-negative ORAI1 (ORAI1-DN). The genetically-modified and control T cells were stimulated (or not stimulated) with antigen in vitro, before they were transferred into a living mouse. Homing and migration behavior of the T cells in the spleen and the LN of the mouse were investigated in situ. While naïve T cells with impaired CRAC function (proven in vitro) showed normal homing into LN, effector T cells, expressing ORAI1-DN were not able to normally approach LN. Motility in LN and spleen of STIM-deficient and ORAI1-DN expressing T cells under steady-state conditions was reduced compared to control (wild type) T cells. In the spleen, it was shown that arrest of CRAC-defective effector T cells was delayed by several minutes compared to control and naïve T cells. The authors showed that TCR stimulation by antigens resulted in elevation of [Ca2+]int and motility

arrest; both were clearly correlated. The in vivo results could not be confirmed in in vitro experiments. In specially prepared planar bilayers, CRAC-defective and control T cells showed the same motility behavior and arrest. The authors conclude [79]: “This suggests that [Ca2+]int elevation-induced arrest in response to TCR activation is especially important in the tissue microenvironment where signals that drive migration may compete with TCR-induced stopping. CRAC channel mediated acute arrest of T cells may play an important role early in immune responses during antigen recognition and priming of T cells. Delayed arrest may cause effector T cells to read-through important APCs resulting in impaired immunity to infection.” To analyze the chronology and signaling pathways of T cell maturation in murine LN in vivo in more detail, Le Borgne et al. [80] developed a knock-in mouse which expresses a mCameleon FRET-based Ca2+ reporter. The reporter was shown to reliably monitor [Ca2+]int in the range of 50 nM to 2 μM, which covers the physiological [Ca2+] levels in T cells. After having confirmed – by comparison with control cells in vitro – that the genetical manipulation of the cells had not altered their “normal” behavior, the researchers measured Ca2+ flux both, in vitro and in explanted LN using Two-photon microscopy. The authors analyzed [Ca2+]int in phase I of T cell differentiation as defined by Mempel et al. [15] (see Introduction). In explanted LN it was found that during phase I, T cells showed substantial changes in motility and “weak Ca2+ fluxes”. The amount of antigen per transiently-contacted DC proved to be directly related to the duration of the contact. The authors suggested that Ca2+ signals accumulated due to multiple contacts of (still naïve) T cells with DC, and that these are the signal for phase II initiation [80]. Comparing experiments in explanted LN with respective in vitro measurements, substantial differences became evident. In vitro, T cells explanted from spleen or LN of mCameleon- transgenic mice, when confronted with APC (here specially prepared bone-marrow-derived macrophages), immediately formed stable contacts. In parallel, [Ca2+]int levels often remained high over hours causing sustained Ca2+ plateaus. Less frequently, transient [Ca2+]int elevations were observed, and rarely irregular Ca2+ oscillations/transitions. Under in vivo conditions, [Ca2+]int showed always sustained elevations with occasional spikes independent of the dose of ovalbumin antigen. T cells with strong contacts more often displayed sustained Ca2+ patterns compared to T cells with “softer” (shorter) contacts which often showed oscillating patterns. Surprisingly, when antigen-presenting DC were present, but not in contact with naïve T cells in the same LN, velocity of migrating T cells decreased, and [Ca2+]int levels increased [80]. Thestrup et al. [23] designed new troponin C-based GECIs, called Twitch, with improved properties for ratiometric Ca2+ measurements. They used different Twitch sensors to analyze [Ca2+]int in vivo in T cells. Antigen-specific T cells were prepared from OT-II mice expressing Twitch-1 or Twitch-2B, and were transferred into wild-type mice, where they appeared in LN. In vivo measurements of T cells by Twophoton microscopy in the absence of antigen revealed low [Ca2+]int and high motility for T cells in both, Twitch-1 or Twitch-2B OT-II mice. Upon injection of the cognate ovalbumin peptide antigen into the mouse, [Ca2+]int levels increased in both sensor-expressing T cell subtypes, and motility decreased. It turned out that with the sensor Twitch2B oscillatory dynamics of [Ca2+]int changes in the antigen-stimulated T cells could be better resolved than with Twitch-1. With the here described in vivo experiments it was convincingly shown that T cells reduce migration velocity and eventually arrest upon antigen stimulation and that this behavior is exactly paralleled by an elevation of [Ca2+]int. Patterns of [Ca2+]int range from sustained signals to irregular Ca2+ oscillations/transitions. Finally, Dong et al. [81] employed human and mouse T cells to study Orai1-dependent T cell motility during the early phase of differentiation. This paper was included in the present section of our review, although no focal T cell stimulation through an IS was used, and consequently, measured data will not be considered in our final evaluation. 88

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Fura-2-loaded T cells. The authors report [Ca2+]int rises up to several hundreds nM from a baseline of 50–100 nM. The responses were mostly oscillatory with stable frequencies of typically one per 100 s at 35 °C and often superimposed on elevated Ca2+ plateaus [86]. The same authors, using the same methods, followed up their initial paper by defining different phases of T cell shape changes and [Ca2+]int signals induced by antigen presentation [87]. Initial deformation of T cells was not accompanied by [Ca2+]int changes (phase I) but resulted finally in Ca2+ store depletion initiating the Ca2+ influx for phases II and III. Phases II and III were thus accompanied by large [Ca2+]int changes: during phase II, T cells rounded up which was paralleled by oscillatory [Ca2+]int patterns. Fluctuations were irregular consisting of short-term Ca2+ plateaus with oscillations or transitions sometimes superimposed on the plateau and sometimes not. The authors also concluded that Ca2+ signals were global and not different at or away from the IS and that the large Ca2+ entry during phase II was intimately connected to the loss of T cell motility, which was still present in phase I. Phase III of the antigen recognition was characterized by new deformations of the T cells and accompanied by a global more steady [Ca2+]int rise. In contrast to the first two phases, phase III could not be mimicked by any non-focal “artificial” T cell stimulation, for instance with thapsigargin [87]. Finally Donnadieu et al. report that low-level TCR stimulation can elicit T cell proliferation at very low [Ca2+]int < 400 nM and that Ca2+ oscillations show no advantage against a simple sustained [Ca2+]int rise [88]. Negulesu et al. [89] worked with a murine HEL-restricted CD4+ T cell clone stimulated by MHC-II-restricted B cells to analyze [Ca2+]int signals after IS formation. The authors used Fura-2PE, a derivative of Fura-2 being more resistant to cellular export and to enrichment in cell organelles [70]. They found that stable T cell-APC contacts correlated well with sustained [Ca2+]int rises which also inhibited T cell motility. Large and frequent oscillatory [Ca2+]int signals induced stable contacts whereas smaller infrequent oscillations correlated with unstable T cellAPC contacts. Wülfing et al. [90] analyzed [Ca2+]int in clonal murine CD4+ T cells stimulated by peptide-loaded I-Ek-transfected CHO cells. The authors observed [Ca2+]int plateaus of varying amplitude depending on stimulation strength as well as transient and partial [Ca2+]int signals. “Partial” in this case was defined as a transition-like behavior of [Ca2+]int between two different states which was mostly irregular and only sometimes showed a distinct frequency resembling Ca2+ oscillations. Zweifach’s group analyzed [Ca2+]int signals in different cytotoxic Tcell lines (AJY CTL and TALL-104) [91,92]. [Ca2+]int measurements were performed in Fura-2-loaded CTL stimulated by antigen-loaded APC (i.e. Raji B cells). The authors observed sustained Ca2+ increases as well as regular or irregular [Ca2+]int changes from a baseline of about 60 nM. Amplitudes of [Ca2+]int elevations were on average around 250 nM with occasionally higher oscillations or individual spikes [91,92]. Measurements in primary human T cells are rare. [Ca2+]int was analyzed in primary human CD4+ T lymphocytes following stimulation by antibody-coated beads by Schwarz et al. [93]. As expected, the authors found a strong dependence of [Ca2+]int on the magnitude of Ca2+ entry. [Ca2+]int increases were often sustained with abrupt transitions between different [Ca2+]int values but no regular Ca2+ oscillations. [Ca2+]int was around 50 nM under resting conditions and increased to values in the higher nanomolar range depending on CRAC channel activity. The Ca2+ dependence of proliferation and apoptosis was also determined. Very low [Ca2+]int increases in the range of 150–300 nM were sufficient to stimulate strong proliferation consistent with previous measurements involving the CRAC channel blocker BTP2 [94]. However, if a [Ca2+]int transient did not reach 90 nM, proliferation was unlikely to occur. Under these conditions, apoptosis of T cells dominated as measured by caspase3 and 7 activities. Thus, low [Ca2+]int signals can in principle be decisive for the fate of T cell clones [93]. The

However, the methodical approach of the study deserves attention. The authors compared in vivo experiments in LN of immune-compromized mice with in vitro studies using microchambers. For both types of experiments, migration behavior and spontaneous [Ca2+]int signals of dominant-negative eGFP-tagged Orai1-E106 A mutant and control human T cells were investigated. The genetically encoded ratiometric Ca2+ sensor Salsa6f (fused from GCaMP6f and tdTomato) was used in the in vitro measurements with transiently transfected primary human T cells; for the in vivo experiments, transgenic mice, expressing Salsa6f homozygously in CD4+ T cells [22], were employed. The main results concerning the importance of Orai1 channel function for T cell motility of the study by Dong et al. are, in principle, similar to those from an earlier report by Greenberg et al. [82]. In addition, it was shown by Dong et al. that Orai1-mutant T cells migrated with higher average velocities and bridged longer distances per time than control cells [81]. This was not because mutant cells moved faster than control cells, but because their directional persistence was increased and they showed fewer migration arrests at rarer intervals than controls. By mimicking essential environmental conditions of the LN in the “migration microchannel assay”, and assessing migration behavior of Orai1-mutant and control T cells in vivo, the authors achieved similar results as in intact LN. As found in vivo, mutant T cells in vitro showed fewer migration arrests at rarer intervals than controls. Dong et al. [81] concluded that functional Orai1 channels regulate arrest phases during migration of T cells and thus decrease overall motility. Furthermore, T cells, in which [Ca2+]int was elevated in a sustained manner, migrated with markedly lower velocity than cells with baseline levels. The authors report that “highly motile T cells always exhibited baseline [Ca2+]int levels, while elevated Ca2+ levels were only found in slower or arrested T cells”. Migration arrest was observed when intrinsic Ca2+ signals (“peaks”) lasted for ≥ 30 s. Interestingly, even when MHC-I and II complexes were blocked by antibodies in CD4+-Salsa6f mice, oscillating Ca2+ signal patterns in the T cells were still present, albeit reduced. In summary, it can be concluded that in vitro experiments, when conducted in the proper environment, can be compared to in vivo studies within intact LN. 2.4. [Ca2+] measurements in primary T lymphocytes and T cell lines in vitro To allow a meaningful comparison of in vitro to in vivo data, we will focus almost exclusively on studies with [Ca2+]int measurements in primary T lymphocytes stimulated focally through an immunological synapse (IS) (Fig. 1) as focal stimulation was shown to differ from nonfocal stimulation regarding Ca2+ signals [5,83]. Whereas antigen-specific transgenic mice exist to allow focal stimulation through an IS with APC, for instance for ovalbumin peptide antigens [84], the situation is more complicated for human T lymphocytes. Technically easy, yet “artificial” is the focal stimulation of T cells with antibody-coated beads, which allow activation of the CD3/TCR complex and CD28 costimulatory receptors (Fig. 1C). More physiological is the stimulation through APC, linking for instance MHC-II and TCR through a “super antigen” (i.e. Staphylococcal enterotoxin A, SEA) (Fig. 1B), which works for both CD4+ and CD8+ lymphocytes. Finally antigen-specific clones can be generated, allowing in vitro stimulation through mature DC (Fig. 1A). The last method is technically challenging and clone generation is time-consuming and expensive. The first [Ca2+]int measurements following antigen-specific stimulation by APC, that we found in the literature, was carried out in a cloned bovine T cell hybridoma. Shapiro et al. [85] reported antigenspecific [Ca2+]int increases within minutes of APC application. The first more detailed study on the kinetics of [Ca2+]int signals by APC was published by Donnadieu et al. [86]. They used a DT4-specific human CD4+ T cell clone called P28D, which could be stimulated by murine fibroblasts transfected with the proper MHC-II receptor. Single T cell-APC contacts were analyzed and [Ca2+]int was determined in 89

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amplitudes of sustained [Ca2+]int elevations can differ for human CD4+ T cell subsets stimulated by antibody-coated beads as described by Kircher et al. [95]. They found that Treg cells showed significantly higher [Ca2+]int than Th0, Th1, Th2 and Th17 subtypes, the latter of which differed only slightly in the [Ca2+]int phenotype among each other. Finally, Zhou et al. [96] measured [Ca2+]int in primary human CTL stimulated by SEA antigen-loaded Raji B cells. They found that IS formation between CTL and Raji B cells induced sustained [Ca2+]int increases to about 100–600 nM depending on CRAC channel activity. The authors correlated these [Ca2+]int signals to the cytotoxic activity of the CTL and surprisingly found that rather small [Ca2+]int signals in the range of 122–334 nM were optimal for killing of Raji cells [96].

oscillations (Fig. 3C, E). Usually, under weak stimulatory conditions, Ca2+ oscillations do not show a distinct frequency, and could thus also be defined as Ca2+ transitions. Whether low avidity (with high affinity TCR) induces weaker or stronger Ca2+ signals, is not evident from the available data (Fig. 3B). Summarizing in vivo and in vitro conditions, all kinds of Ca2+ signals as shown in Fig. 3 are observed. The hitherto existing quantity of reports is however not yet sufficient to give a final answer whether in vivo and in vitro [Ca2+]int data compare well. The major results, however, appear to be similar for primary T cells. All depicted Ca2+ patterns were observed in vivo and in vitro. Elevation of [Ca2+]int decreases T cell motility. [Ca2+]int signals appear to largely depend on Ca2+ influx through Orai channels. Whether the in vivo [Ca2+]int data from murine T cells compare well to the in vitro data from human T cells cannot be definitely answered. Patterns appear to be similar but quantification is not possible considering the small amount of published data. The most obvious difference we noticed between in vivo and in vitro data, are the dynamics of T cell-APC interactions. Under in vitro conditions, long-lasting T cell-APC contacts are formed immediately after encountering; in vivo, cells show several transient interactions before they stay together in strong and long-lasting contacts. This difference may be caused by the lack of (essential) environmental factors in invitro studies and, additionally by the artificially low temperatures used during in vitro experiments (usually RT to 35 °C), which may change among others cytoskeletal T cell dynamics.

3. Comparing in vivo and in vitro T lymphocyte [Ca2+] signals We have summarized [Ca2+]int signals in T lymphocytes following focal stimulation by an IS with APC (Fig. 1). Since [Ca2+]int measurements from non-focal stimulation (like thapsigargin, a combination of Ca2+ ionophores and phorbolesters, lectins or dissolved antibodies against the TCR) or spontaneously occurring Ca2+ signals differ from the ones stimulated by APC, we have not included these studies in this review. For detailed overviews please compare [1–10]. Data compared here are mostly from primary murine T cells, murine T cell lines, or from primary human T lymphocytes. We have summarized the in our opinion most relevant in vivo studies [71,75,76,97], ex vivo studies from explanted LN [23,33,77–80] and in vitro data [86,87,89–93,96]. Of course, our selection of reports may not be complete as we might have overlooked important papers, but it should allow a valid comparison and evaluation of the current state of knowledge about [Ca2+]int signals in T cells. From the literature it is quite clear that antigen potency, defined by antigen affinity and avidity is a crucial factor to determine [Ca2+]int signals. In several of the above cited reports it was shown that strong antigen avidity induces large sustained Ca2+ signals, oscillations with high amplitudes or a combination of both (Fig. 3A, D). Low avidity (with low affinity TCR) more likely causes small and infrequent Ca2+ spikes, transient Ca2+ signals or Ca2+ transitions or irregular

4. Conclusions and perspectives In vivo and in vitro [Ca2+]int measurements of primary murine and human T lymphocytes stimulated focally through an immunological synapse with antigen-presenting cells reveal common Ca2+ patterns. These include sustained Ca2+ signals in the nanomolar range but also Ca2+ oscillations, which are, however, in particular under in vivo conditions relatively irregular. They should maybe better referred to as Ca2+ spikes or Ca2+ transitions between different sustained [Ca2+]int values. While the number of studies is still limited, the general features of [Ca2+]int signals are conserved between in vivo and in vitro conditions. However, for a definite quantitative comparison, many more well

Fig. 3. Comprehensive scheme on focal stimulation of T cells by APC and the dependence of [Ca2+]int responses on stimulation strength. (A–C) The strength is determined by a high/low affinity (binding strength) of a T cell receptor (TCR) for a special antigen and by the avidity, which describes the sum of all affinities. (D) Schematic [Ca2+]int traces (high avidity combined with high or low affinity TCR) correspond to “strong” stimulation of T cells, as depicted in A. (E) [Ca2+]int signals due to “weak” stimulation (low affinity and low avidity TCR) as shown in C. Abbreviations are as in Fig. 2. 90

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Fig. 4. Comparison of model systems to study [Ca2+]int signals in T lymphocytes. Primary cells in the second column refer to human and mouse. Currently used methods (and possible ones in the future) applied for in vitro, in vivo and ex vivo systems are depicted. The listed advantages and disadvantages of the respective system may determine the suitability for transfer of results to the human system.

transferred into more physiological 3D environments analyzed by lightsheet microscopy (Fig. 4) [101,102]. Again, CTL are a good model system because their cytotoxic function can be correlated with [Ca2+]int [96]. For these correlations, simple kinetic real-time cytotoxicity assays [103] or more sophisticated assays that allow discrimination between target cell apoptosis or necrosis [104] are needed. As already mentioned in the previous chapter it is mandatory to control antigen potency of APC when correlating quantitative single cell [Ca2+]int with the cytotoxic potency of the respective CTL. Finally we want to highlight that targeting of genetically encoded Ca2+ indicators to organelles allows in vivo subcellular Ca2+ imaging. As in most cell types, local Ca2+ signals likely play a role for T cell function. They have been reported at the immune synapse [105] and during the initial Ca2+ release phase from ER stores [106], however the functional importance of local Ca2+ signaling is just starting to emerge. Nuclear translocation of the transcription factor NFAT might be promising process to be controlled by local [Ca2+]int as recently shown in HEK cells [107]. A direct link between NFAT and cytotoxicity has also been recently reported [108], again pointing to CTL as a good model system to compare in vivo and in vitro [Ca2+]int and its correlation with single cell cytotoxicity. We conclude by stressing that in vivo [Ca2+]int measurements in mice should be paralleled by in vitro [Ca2+]int measurements in primary human T cells under conditions mimicking physiological conditions as good as possible (i.e. 37 °C, three dimensional matrix) with a particular focus on controlling the avidity and affinity of cognate antigenic stimulation by APC.

defined experiments are needed. In particular, it would be essential to find a common definition for the term “antigen potency”, which is not sufficiently explained in most studies. Since antigen affinity and avidity were shown to greatly influence Ca2+ signaling in T cells (Fig. 3), a clear definition and a measure for these parameters are required. Of course, this is very challenging, in particular under in vivo conditions. Due to the high physiological relevance of studies in living vertebrates for human physiology, which is great advantage over all other methods (Fig. 4), every effort should be taken to generate long in vivo [Ca2+]int measurements in T cells of living animals and link them to specific functions. The development of different, partly ratiometric genetically encoded Ca2+ indicators including calmodulin-based GCamP6 sensors [21,22] and troponin-based Twitch sensors [23] should facilitate these measurements in transgenic mice (Fig. 4). On the functional site, the analysis of CD4+ T cells is mostly limited to correlate [Ca2+]int with motility and contact times with APC during IS formation. This is of course not a direct measure of T cell differentiation. Linking [Ca2+]int with specific T cell functions may be facilitated by investigating cytotoxic T lymphocytes. In CTL, [Ca2+]int can be easily correlated with the cytotoxic efficiency of CTL against their antigen-presenting target cells. A first step in the direction was done by Halle et al. [97] who investigated CTL killing of virus-infected target cells. [Ca2+]int was monitored by GCaMP6, which was, however, not expressed in CTL but in target cells to observe heterogeneity of target cell-CTL contacts. To study Ca2+ dependent CTL cytotoxicity in vivo Kim et al. [98] recently applied optogenetic tools. They used CTL, expressing CatCh, a new variant of channelrhodopsin, which has a very high light sensitivity and accelerates Ca2+ membrane permeability. The measurements showed that CTL increase their cytotoxic functions against tumor cells and overcome Treg-mediated suppression by light-activated Ca2+ influx [98]. Given the disadvantage of technical limitations of in vivo [Ca2+]int measurements in T cells (Fig. 4) and the many reported differences between murine and human immunity [99,100], a focus on in vitro [Ca2+]int measurements in primary human T cells is also highly desirable. Measurements are relatively easy and can also easily be

Acknowledgements We are very thankful to Sandra Janku for providing language help. This work was supported by Deutsche Forschungsgemeinschaft (DFG), in particular by the collaborative research centers SFB 894 and SFB 1027.

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