LFA-1-dependent Ca2+ Entry following Suboptimal T Cell Receptor Triggering Proceeds without Mobilization of Intracellular Ca2+*

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-γ1 (PLC-γ1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-γ1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-γ1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

The T cell encounter with antigen-presenting cells (APCs) 2 carrying cognate peptide in the context of major histocompatibility complex (MHC) is followed by an increase in cytosolic Ca 2ϩ ion concentration ([Ca 2ϩ ]) by entry of extracellular Ca 2ϩ (1). The extracellular Ca 2ϩ entry is key for a myriad of physiological changes leading to cell cycle progression and development of effector functions of T cells. Downstream signaling events requiring an influx of extracellular Ca 2ϩ involve activation of calcineurin/NF-AT and Ras/mitogen-activated protein kinase (MAPK) signaling pathways together resulting in transcriptional activation of multiple genes, including interleukin-2 (2,3).
A well known Ca 2ϩ -signaling mechanism in T cells is store-operated Ca 2ϩ entry (SOCE). According to that mechanism, phospholipase C-␥1 (PLC-␥1) activated by T cell receptor (TCR) stimulation catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5trisphosphate (IP 3 ), which binds to the IP 3 receptor in the endoplasmic reticulum to induce release (mobilization) of Ca 2ϩ from the endoplasmic reticulum. As a result, Ca 2ϩ channel in plasma membrane opens to allow entry of extracellular Ca 2ϩ . Recent advances in the field entail identification of specific proteins involved in the process such as STIM-1, a Ca 2ϩ sensor in endoplasmic reticulum, and ORAI-1, a Ca 2ϩ channel in plasma membrane (4).
LFA-1 (lymphocyte function-associated antigen-1), an integrin composed of ␣L (CD11a) and ␤ 2 (CD18) subunits, plays multiple roles in various stages of T cell immune responses, i.e. in activation of resting T cells, migration of activated effector T cells to the site of infection, and execution of their effector functions (5,6). LFA-1 acts as both an adhesion and a signaling molecule so that interaction of LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), not only facilitates firm contact between T cell and APC but also induces intracellular signaling events (7).
The importance of LFA-1 in Ca 2ϩ signaling has been identified. Earlier studies showed that engagement of LFA-1 resulted in prolonged IP 3 production and the sustained increase of intracellular [Ca 2ϩ ] as well as stronger PLC-␥1 activation (8,9). It was also shown that a specific motif (NPXF) in the intracellular domain of the CD18 subunit was responsible for the action of LFA-1 (10). In addition, a study by Bachmann et al. (11) showed that costimulation by LFA-1 lowered the threshold level of cognate peptide-MHC complex (pMHC) expression required for induction of extracellular Ca 2ϩ entry during the T/APC interaction. It was also suggested that LFA-1 facilitated entry of extracellular Ca 2ϩ by promoting the formation of immunological synapse by which production of IP 3 could be amplified and stabilized (12).
We have been using nanometric membrane vesicles prepared from the purified plasma membrane fraction of Drosophila cells engineered to express L d class I MHC plus B7-1 and  ICAM-1, along with 2C TCR transgenic T cells, for studying  membrane-proximal TCR signaling events leading to LFA-1 activation (13,14). In this study, we investigated Ca 2ϩ signaling of 2C T cells being cultured with the plasma membrane-derived membrane vesicles (pMVs), and we present data revealing that LFA-1-dependent Ca 2ϩ entry, manifest in T cells undergoing the activation process after suboptimal TCR stimulation, proceeds via a mechanism separate from SOCE.
Preparation of pMVs-The pMVs were prepared as described by Goldberg and Kornfeld (16) with modifications. In brief, Drosophila APCs induced to express transfected mouse immunomolecules were washed and resuspended in buffer A (15 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 10 mM Hepes, pH 7.5) containing a mixture of protease inhibitors (Sigma) and lysed with Contes homogenizer (loose-fitting pestle). The cell lysate was spun down at low centrifugal force (1500 ϫ g) to remove unlysed cells and nuclei. The supernatant was recovered and mixed with 0.5 volume of buffer B (375 mM KCl, 22.5 mM MgCl 2 , 1 mM dithiothreitol, 220 mM Hepes, pH 7.5) containing protease inhibitors and subjected to ultracentrifugation (1 h at 50,000 ϫ g at 4°C) to collect the crude membrane particles. The resulting pellet was resuspended in buffer C (40 mM Tris⅐Cl, 10 mM KCl, 10 mM NaCl, 0.2 mM EDTA, pH 7.2) and finely dispersed using Contes homogenizer (tight-fitting pestle). Resuspended membrane vesicles were fractionated by sucrose gradient ultracentrifugation, 200,000 ϫ g for 2 h at 0°C. Membrane vesicles derived from plasma membrane fractions were taken from the interface of 20:35% sucrose layers, aliquoted, and stored at Ϫ80°C. The amount of pMVs was determined by measuring the protein concentration in the sample.
Western Blot Analysis-2C T cells cultured with peptideloaded pMVs were washed with ice-cold 1ϫ phosphate-buffered saline and lysed in a cell lysis buffer (50 mM Tris⅐HCl, 150 mM NaCl, 1% Igepal CA-630, pH 7.5) containing a mixture of protease inhibitors (Sigma) followed by sonication for further homogenization. Equal amounts of lysates (determined by the protein concentration) were separated by SDS-PAGE and subjected to Western blotting as described (17). Proteins of interest were visualized by ECL reagent (Pierce).
Kinetic Flow Cytometric Analysis of Cytosolic [Ca 2ϩ ]-Kinetic analysis of changes in cytosolic [Ca 2ϩ ] was performed as described (18). Briefly, 700 l of purified CD8 ϩ 2C T cells (2 ϫ 10 6 cells/ml) were loaded with 3 M Indo-1/Am (Invitrogen) for 50 min at 37°C followed by thorough washing with ice-cold 1ϫ phosphate-buffered saline and resuspended in normal Dulbecco's modified Eagle's medium (catalog number 21041, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum or Ca 2ϩ -free Dulbecco's modified Eagle's medium (catalog number 21068, Invitrogen) supplemented with 10% heat-inactivated dialyzed fetal bovine serum. Seven hundred microliters of Indo-1-loaded 2C T cells (2 ϫ 10 6 /ml) were prewarmed for 15 min at 37°C and mixed with 70 l of peptideloaded pMVs (1 mg/ml). Flow cytometric analysis began 60 s before addition of pMVs and continued for 720 s afterward; flow cytometric analysis was carried out with an LSR II flow cytometer equipped with a temperature control device. Changes in the concentration of intracellular Ca 2ϩ were determined measuring changes in the ratio of fluorescence intensities of Indo-1-loaded T cells detected by two separate photomultiplier tubes with bandpath filters of 440/40 nm (violet) and 530/30 nm (blue), respectively; Indo-1 was excited by a 351-nm UV laser.
Phosphorylation of Akt at Thr 308 occurred rapidly upon culture of 2C T cells with the pMVs loaded with QL9 (1 M) peptide. The level of the Akt phosphorylation reached near maximum within 5 min of culture and was sustained through 30 min (Fig. 1A, left). Phosphorylation of PLC-␥1 at Tyr 783 was also detected within 5 min of the culture, but the level of the phosphorylation increased gradually as the culture time extended (Fig. 1B, left).
Increase of Cytosolic [Ca 2ϩ ] during Culture of 2C T Cells with the pMVs-Observation that the tyrosine phosphorylation of PLC-␥1 progressed quickly during culture of 2C T cells with the pMVs loaded with QL9 peptide prompted us to examine Ca 2ϩ signaling during the culture.
Wild type 2C T cells being cultured with the QL9 (1 M)-loaded L d B7-1ICAM-1 pMVs showed a prominent increase in cytosolic [Ca 2ϩ ] (Fig. 2). The increase became evident ϳ2.5 min after mixing of 2C T cells with the pMVs and continued through the culture up to 12 min. mAb blocking of CD28/B7-1 interaction slightly reduced the extent of Ca 2ϩ increase (Fig. 2, left). In contrast, mAb blocking of LFA-1/ICAM-1 interaction almost completely abolished the Ca 2ϩ increase. Importance of LFA-1/ICAM-1 interaction in the cytosolic Ca 2ϩ increase was confirmed by experiments using 2C. LFA-1 Ϫ/Ϫ T cells, in which no measurable Ca 2ϩ increase was observed. Consistent with the result obtained with 2C T cells treated with anti-CD28 mAb, 2C.CD28 Ϫ/Ϫ T cells showed conspicuous increase in cytosolic [Ca 2ϩ ] during the culture (Fig. 2).
Lack of Intracellular Ca 2ϩ Mobilization in 2C T Cells Cultured with L d B7-1ICAM-1 pMVs-According to the mechanism of SOCE, entry of extracellular Ca 2ϩ resulting in a surge in cytosolic [Ca 2ϩ ] is preceded by mobilization of Ca 2ϩ from intracellular Ca 2ϩ store following PLC-␥1 activation by TCR triggering (2). We thus examined whether the increase in cytosolic [Ca 2ϩ ] observed during culture of 2C T cells with QL9-loaded L d B7-1ICAM-1 pMVs was accompanied by mobilization of intracellular Ca 2ϩ by culturing the cells in a Ca 2ϩ -free medium (Fig. 3). Surprisingly, no change in cytosolic [Ca 2ϩ ] was detected during the culture (Fig. 3A, right). Note that strong mobilization of intracellular Ca 2ϩ was detected after treatment of 2C T cells with thapsigargin, a well known intracellular Ca 2ϩ mobilizer, both in the Ca 2ϩ -containing and -free media (Fig. 3A, left) (21).
Detection of Intracellular Ca 2ϩ Mobilization in 2C T Cells Cultured with pMVs Expressing Higher Level of L d -The level of L d expression in L d B7-1ICAM-1 Drosophila APCs is modest (supplemental Fig. S2). Thus, it was suggested that the level of L d /QL9 expression in L d B7-1ICAM-1 pMVs was too low to trigger TCR signaling strong enough to induce mobilization of intracellular Ca 2ϩ in primary resting T cells such as 2C transgenic T cells. To test the idea, we used pMVs prepared from Drosophila APCs expressing higher levels of L d , namely pMVs prepared from two separate Drosophila APC clones expressing L d alone (L d ) and higher level of L d plus B7-1 plus ICAM-1 (L d(high) B7-1ICAM-1) (supplemental Fig. S2).
2C T cells cultured with L d pMVs loaded with QL9 peptide (1 M) in Ca 2ϩ -containing medium showed a rapid increase in intracellular [Ca 2ϩ ], which became evident less than a minute after addition of the pMVs (Fig. 3B, left). Different from the Ca 2ϩ increase by L d B7-1ICAM-1 pMVS, the Ca 2ϩ increase by L d pMVs did not proceed progressively; instead, after an initial spike, the [Ca 2ϩ ] decreased slightly and was sustained at a steady level throughout the culture. Consequently, at the end of the culture, the cytosolic [Ca 2ϩ ] in 2C T cells cultured with QL9-loaded L d pMVs was considerably lower than that in 2C T   AUGUST 14, 2009 • VOLUME 284 • NUMBER 33

JOURNAL OF BIOLOGICAL CHEMISTRY 22151
cells cultured with the QL9-loaded L d B7-1ICAM-1 pMVs (Fig.  3). Nevertheless, a transient but significant increase in cytosolic [Ca 2ϩ ] was detected in 2C T cells cultured with QL9-loaded L d pMVs in Ca 2ϩ -free medium (Fig. 3B, left).
2C T cells cultured with L d(high) B7-1ICAM-1 pMVs loaded with QL9 (1 M) peptide in Ca 2ϩ -containing medium showed a rapid and progressive increase in the concentration of cytosolic Ca 2ϩ (Fig. 3B, right). The [Ca 2ϩ ] measured after 12 min of culture with QL9-loaded L d(high) B7-1ICAM-1 pMVs was higher than that measured after culture with L d B7-1ICAM-1 pMVs, but the difference was marginal. As with QL9-loaded L d pMVs, rapid and transient increase in cytosolic [Ca 2ϩ ] was detected when 2C T cells were cultured with QL9loaded L d(high) B7-1ICAM-1 pMVs in Ca 2ϩ -free medium (Fig. 3B, right).
Level of Cognate pMHC Expression Versus LFA-1 Dependence of Ca 2ϩ Signaling-Next, we examined an effect of concentration of QL9 peptide loaded to L d(high) B7-1ICAM-1 pMVs on both entry of extracellular Ca 2ϩ and mobilization of intracellular Ca 2ϩ observed in Ca 2ϩ -containing and Ca 2ϩ -free media, respectively (Fig. 4). When wild type 2C T cells were cultured with the pMVs loaded with QL9 peptide at 200 and 40 nM, respectively, both mobilization of intracellular Ca 2ϩ and influx of extracellular Ca 2ϩ were readily detected. When the pMVs were loaded with the peptide at 8 nM, a considerable increase in cytosolic [Ca 2ϩ ] was detected when the T cells were cultured in Ca 2ϩ -containing medium, although no measurable change was monitored when cultured in Ca 2ϩfree medium. When the pMVs were loaded with the peptide at a further lower concentration (1.6 nM), no Ca 2ϩ increase was detected in either medium (Fig. 4, left).
When 2C.LFA-1 Ϫ/Ϫ T cells were used in the experiments (Fig. 4, right), different results were obtained. As with wild type 2C T cells, the mutant T cells being cultured with L d(high) B7-1ICAM-1 pMVs loaded with QL9 peptide at higher concentrations (200 and 40 nM, respectively) displayed both influx of extracellular Ca 2ϩ and mobilization of intracellular Ca 2ϩ . The pattern of Ca 2ϩ increase in the mutant 2C T cells cultured in Ca 2ϩ -containing medium was, however, different from that in wild type 2C T cells and rather resembled that in wild type 2C T cells cultured with QL9-loaded L d pMVs (Fig. 3B). When the same mutant T cells were cultured with L d(high) B7-1ICAM-1 pMVs loaded with the peptide at lower concentrations (8 and 1.6 nM, respectively), they showed no Ca 2ϩ increase in either medium.
Role of PLC-␥1 in LFA-1-dependent Extracellular Ca 2ϩ Entry-We also examined the involvement of key signaling molecules engaged in membrane-proximal TCR signaling, i.e. PTKs, PI3K, and PLC-␥1, in LFA-1-dependent Ca 2ϩ entry using pharmacological agents specifically targeting those molecules. Treatment of wild type 2C T cells with PP2, Ly294002, or U73122, which target PTKs, PI3K, and PLC-␥1, respectively, before culture with L d B7-1ICAM-1 pMVs loaded with QL9 (1 M) peptide strongly inhibited the Ca 2ϩ entry (Fig. 5), indicating a critical role of those signaling molecules in the LFA-1-dependent Ca 2ϩ entry. The importance of PI3K was further sup-   ported by comparable inhibition of the Ca 2ϩ entry by wortmannin (500 nM), another well known PI3K inhibitor. 3

DISCUSSION
Microvesicles prepared from plasma membrane of Drosophila APCs expressing defined mouse immunomolecules of interest (i.e. pMVs) (13,14,22) have several advantageous features in investigation of membrane-proximal signaling mechanisms primed by TCR stimulation. Given that physiological ligands expressed in biological membrane are used for stimulating T cells, experimental results obtained with pMVs may have superior physiological relevance compared with those obtained with receptor-specific mAbs or ligands immobilized on the surface of plastic or magnetic beads. Because of the huge difference in their sizes, separation of T cells from pMVs after culture is simple, and thus instant biochemical analysis of molecular alterations occurring during the culture becomes feasible; when intact cells are used for stimulating T cells, a complicated method is needed to separate T cells from stimulating cells. In addition, kinetic flow cytometric analysis of physiological changes occurring in the early stage of T cell activation, e.g. influx of extracellular Ca 2ϩ , is facilitated.
We initiated this study for investigating contributing roles of CD28/B7-1 and LFA-1/ICAM-1 interactions in a series of molecular changes in the early stage of T cell activation. We have shown before that the sole interaction of 2C TCR with L d -QL9 complex expressed in L d B7-1ICAM-1 pMVs is necessary and sufficient to induce a near maximum level of F-actin polymerization; in that process, CD28 and LFA-1 played little role (14). Different from in F-actin polymerization, both CD28 and LFA-1 took a significant and comparable part in phosphorylation of Akt ( Fig. 1A and supplemental Fig. S1), conforming to their roles in activation of PI3K signaling cascades reported by others (23,24). They also exerted comparable effects on phosphorylation of PLC-␥1 ( Fig. 1B and supplemental Fig. S1). Considering that PLC-␥1 phosphorylation at Tyr 783 is mediated by the Syk family PTK, i.e. ZAP-70 in T cells, one may reason that CD28 and LFA-1 may take part in activation of ZAP-70 (25). Alternatively, it is also plausible that CD28/B7-1 and LFA-1/ICAM-1 interactions make contributions to the phosphorylation of PLC-␥1 via up-regulation of PI3K activity through which more phosphatidylinositol 3,4,5-trisphosphate becomes available. As a result, functionality of signaling molecules containing the PH domain is escalated facilitating formation of signalsome centered by PLC-␥1 and phosphorylation of PLC-␥1 by ZAP-70 (26,27).
In striking contrast to roles in the PLC-␥1 phosphorylation, roles of CD28 and LFA-1 in the increase of cytosolic [Ca 2ϩ ] were clearly distinct (Fig. 2). Considering that phosphorylation of PLC-␥1 at Tyr 783 is reportedly critical for activation of its enzymatic activity (19), those results were intriguing and suggested a novel Ca 2ϩ entry mechanism exerted by LFA-1 other than SOCE. Supporting that notion, the LFA-1-dependent Ca 2ϩ entry proceeded without mobilization of measurable levels of intracellular Ca 2ϩ (Fig. 3A). The lack of intracellular Ca 2ϩ mobilization during culture of 2C T cells with L d B7-1ICAM-1 pMVs was ensured by the result that pre-culture of 2C T cells with the QL9-loaded pMVs in the calcium-free medium did not alter either onset or magnitude of intracellular Ca 2ϩ mobilization by thapsigargin (supplemental Fig. S3).
Studies for SOCE have been routinely conducted following Ab cross-linking of TCRs, which elicits strong Signal 1; the TCR-mediated signaling mechanisms are collectively termed as Signal 1 (28). Reflecting the importance of Signal 1 in mobilization of intracellular Ca 2ϩ , a transient but distinct level of intracellular Ca 2ϩ mobilization was readily detected when 2C T cells were cultured with pMVs expressing an elevated level of L d class I MHC (Fig. 3B). The mobilization of intracellular Ca 2ϩ occurred almost instantly after mixing of 2C T cells with the pMVs and peaked within a minute of the culture. In line with this, an increase in cytosolic [Ca 2ϩ ] in 2C T cells cultured with the same pMVs in Ca 2ϩ -containing medium progressed much more promptly than with L d B7-1ICAM-1 pMVs.
Experiments using wild type and LFA-1 Ϫ/Ϫ 2C T cells cultured with L d(high) B7-1ICAM-1 pMVs loaded with titrated concentrations of QL9 peptide confirmed that significance of the LFA-1/ICAM-1 interaction in extracellular Ca 2ϩ entry was determined by the level of cognate pMHC expression in the pMVs (Fig. 4). Under conditions that mobilization of intracellular Ca 2ϩ was detected, namely when 2C T cells were cultured with the pMVs loaded with high concentrations of QL9 peptide, extracellular Ca 2ϩ entry occurred without involvement of LFA-1/ICAM-1 interaction even though the Ca 2ϩ entry proceeded more progressively in the presence of LFA-1/ICAM-1 interaction. Extracellular Ca 2ϩ entry could still occur under a condition that intracellular Ca 2ϩ mobilization was not detected. In that condition, extracellular Ca 2ϩ entry was totally dependent on LFA-1/ICAM-1 interaction.
It is of interest that LFA-1-dependent Ca 2ϩ entry still relies on activity of PLC-␥1 (Fig. 5) because the level of PLC-␥1 phosphorylation at Tyr 783 showed little correlation with the level of cytosolic Ca 2ϩ increase ( Figs. 1 and 2). PLC-␥1 plays multiple roles in T cell activation. Hydrolysis of phosphatidylinositol 4,5-bisphosphate catalyzed by PLC-␥1 results in production of diacylglycerol (DAG) as well as IP 3 . DAG is imperative for activation of a family of protein kinase C and small GTP-binding proteins such as Rap1 (29). Activation of Rap1 by DAG may hold significant implications in LFA-1-dependent Ca 2ϩ entry because of its role in inside-out signaling for LFA-1 activation (7,30). Thus, inhibition of LFA-1-dependent Ca 2ϩ entry by the PLC-␥1 inhibitor (U73122) could be explained by inhibition of DAG production by which LFA-1 activation process is impeded, and consequently signaling cascades promoted by high affinity/avidity LFA-1/ICAM-1 interaction are silenced (31).
In addition to Ca 2ϩ channels involved in SOCE (e.g. ORAI-1), other types of Ca 2ϩ channels are also expressed in T cells, e.g. L-type Ca 2ϩ channel and transient receptor potential channel (TRPC) (2,3,32). Here we hypothesize that TRPC-mediated Ca 2ϩ entry mechanism may be of special relevance to LFA-1dependent Ca 2ϩ entry for the following reasons. First, TRPCmediated TCR-dependent Ca 2ϩ entry has been revealed in human T cells (33). Second, TRPC-mediated Ca 2ϩ entry has been found to operate without mobilization of Ca 2ϩ from the intracellular Ca 2ϩ store (34). Third, a few TRPCs have been found to be activated by DAG (35). According to recent study by Mor et al. (36), interaction of LFA-1 with ICAM-1 induces strong activation of phospholipase D to produce DAG functioning in the plasma membrane. Together, it appears possible that TCR triggering leads to activation of LFA-1 via inside-out signaling, and consequently, high affinity/avidity interaction of LFA-1 with ICAM-1 results in amplification of DAG production via activation of phospholipase D. As a result, formation of TRPC is facilitated to allow entry of extracellular Ca 2ϩ .
It has been perceived that enhancement of Ca 2ϩ signaling by LFA-1 is attained via strengthening signaling cascades induced by the TCR/pMHC interaction, namely stable and prolonged T/APC contact established by the LFA-1/ICAM-1 interaction allows more sustainable TCR signaling to facilitate activation of the downstream Ca 2ϩ signaling pathway (11). The results of this study appear to contradict that view and instead represent that LFA-1 activated by TCR triggering acts independently to drive extracellular Ca 2ϩ entry via a mechanism separate from SOCE.