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Ca²⁺ Signals in CD4⁺ T Cells during Early Contacts with Antigen-Bearing Dendritic Cells in Lymph Node¹

Sindy H. Wei^*, Olga Safrina^*, Ying Yu^*, Kym R. Garrod^*, Michael D. Cahalan^2,*, and Ian Parker^*,

^* Department of Physiology and Biophysics, Center for Immunology, Department of Neurobiology and Behavior, University of California, Irvine, CA 92697

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
T cell activation by APC requires cytosolic Ca²⁺ ([Ca²⁺]_i) elevation. Using two-photon microscopy, we visualized Ca²⁺ signaling and motility of murine CD4⁺ T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca²⁺ spikes associated with sustained slowing. Inflammation reduced velocities and Ca²⁺ spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca²⁺ spike frequency and elevated basal [Ca²⁺]_i. These Ca²⁺ signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca²⁺]_i and spiking frequency constitute a Ca²⁺ signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To sustain adaptive immunity, a pool of naive CD4⁺ T cells traffic through secondary lymphoid tissues in a relentless search for cognate Ags that have been transported from the periphery to the T zone by soluble transport or by dendritic cell (DC)³ immigrants (1, 2). Under the influence of pathogen-specific "danger signals" that enhance presentation of peptide MHC (pMHC) and costimulation by DCs, recognition of pMHC by Ag-specific TCR results in T cell activation, proliferation, and effector response (3, 4). In vitro studies have shown that the earliest step in this Ag-specific T cell response involves adhesion to DCs and formation of a contact zone, followed rapidly by a TCR signaling cascade that triggers elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) (5, 6, 7, 8, 9). In the absence of Ag, T cells can also adhere to DCs with subsequent formation of contact zones, but the resultant Ca²⁺ signals are infrequent, transient, and weak (7, 10, 11).

Ca²⁺ signals have been studied in T cells using a variety of in vitro preparations, including two-dimensional culture and three-dimensional (3D) collagen gel matrix systems, in response to stimulation by mitogen, soluble ligands, pMHC delivered on lipid bilayers, or APC conjugation. These experiments revealed a heterogeneity of Ca²⁺ signals ranging from transient peaks, through oscillations, to sustained plateaus (7, 9, 12, 13, 14, 15, 16). Ca²⁺ signaling triggers lymphocyte activation by modulating gene transcription through Ca²⁺-dependent regulation of nuclear transcription factors and that in turn shapes the outcome of the immune response (12, 17, 18, 19). Furthermore, variations in [Ca²⁺]_i signal modality and strength have been correlated with acute differences in T cell motility and polarization, as well as differences in stability of the immunological synapse between T cells and APC (9, 20, 21). The diversity in patterns and intensities of Ca²⁺ signaling may be explained in part by differences in the type of stimulation used, the agonist strength of the peptide Ag, the subset and activation state of T cells or cell lines used, and the type and maturation status of the APC.

A significant limitation of these in vitro experiments, however, is that they fail to appropriately replicate the physiological scaffolding and environment in which T cell-DC interactions normally occur. Whereas T cell adhesion to substrates or DCs heightens the sensitivity of Ag detection and enhances downstream signaling from the TCR (22), observations of brief T cell-DC interactions and Ca²⁺ signals within a 3D collagen matrix have led to the proposal that T cell activation requires serial encounters with APCs and signal integration (11). Therefore, experiments performed within intact lymphoid tissue in the context of an acute infection are needed to clarify the modality, strength, and kinetics of Ca²⁺ signals required for a productive T cell response.

Multiphoton microscopy within the lymph node (LN), using both intravital and explant preparations, has delineated the choreography between naive T cells and mature DC populations (both endogenous and exogenous) that commit the T cell to activation (23). The details of these interactions differ among studies, but a common theme involves the sequential development of discrete modes of interactions that represent evolving phases of the immune response (24, 25, 26, 27, 28). However, whereas several reports describe brief, stochastic, and sequential initial encounters with multiple DCs pointing to successive changes in T cells as they integrate information (24, 25, 29, 30, 31), others report rapid arrest and stable interaction of T cells on DC networks near high endothelial venules (HEV) (26). Thus, variations in T cell-DC interaction may also be attributed to DC maturation status (27) or location in the LN (24). Transient and stable interactions are both associated with antigenic stimulation, but it is unclear whether they are involved in information exchange during T cell priming.

Our understanding of how these diverse interactions relate to T cell activation would be greatly enhanced by simultaneous imaging of both Ca²⁺ signals and behavior of mature T cells during Ag recognition. We thus used two-photon microscopy to measure Ca²⁺-dependent signals from the ratiometric fluorescent indicator 1-[2-amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Indo-1) during early Ag encounter of naive CD4⁺ T lymphocytes inside LN explants. We sought to differentiate between types of Ca²⁺ signaling occurring during the basal (preimmune) state, cytokine (cyt)-induced inflammatory conditions, and Ag-specific immune responses and to directly examine Ca²⁺ signals elicited during T cell-DC interaction, so as to gain insight on how DCs may commit T cells to activate.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Male BALB/c and DO11.10 (OVA_323–339 peptide-specific CD4 TCR-transgenic) mice 4–10 wk of age were purchased from The Jackson Laboratory and used in accordance with protocols approved by the animal care committee (Institutional Animal Care and Use Committee).

Immunization and labeling of endogenous APC

BALB/c recipient mice were immunized beneath the dorsal surface of both ears (25 µl each) and s.c. at the scruff (50 µl), with a mixture of Ag (100 or 300 µg OVA) and cyt (5 µg of FMS-like tyrosine kinase 3 ligand and 2 µg of TNF-) in an adjuvant of 1.3% aluminum hydroxide gel adjuvant (alum, Alhydrogel; Superfos Biosector). Control mice were sham immunized with cyt and alum. In the nonimmunized controls, PBS alone was injected at the immunization sites. To label dermal DCs that traveled to draining superficial cervical LN from the injection sites, 0.5–1 µg of CFSE (Molecular Probes) was added to the immunization mixture. A similar protocol has been shown to generate a robust immune response (25), which we confirmed by showing the up-regulation of CD69 on the T cell surface within 2 and 24 h, more than seven rounds of proliferation over 3–5 days and a significant delayed-type hypersensitivity (DTH) response with Ag rechallenge (data not shown). Moreover, the addition of cyt to the immunization protocol for the imaging experiments did not alter the CD69, proliferative, or DTH profiles compared with Ag and alum alone.

Bone marrow-derived DCs (BMDCs)

Bone marrow cells from the tibia and femur of BALB/c mice were cultured in supplemented medium (RPMI 1640 or IMDM), 10% FCS, and 20 ng/ml recombinant mouse GM-CSF and IL-4 (BD Pharmingen and R&D Systems, respectively). Cells were passaged as needed and used at day 8 or day 11. In some preparations, 30 ng/ml stem cell factor was added on day 0 and did not result in differences in the up-regulation of maturation markers assayed by flow cytometry. OVA was filtered and added to a portion of the culture at 1 mg/ml for 10 h. Maturation of all cells was achieved by addition of LPS (0.5 µg/ml) to the culture medium for 16 h. Loosely adherent cells were harvested from culture, washed twice to remove Ag and cyt, stained with CellTracker Red CMTPX (Molecular Probes), and injected at multiple s.c. sites draining to both popliteal and inguinal nodes. Control cells without exposure to OVA were injected into the contralateral limb. The number of cells injected ranged between 0.2 and 2 x 10⁶ and did not appreciably affect the density of BMDCs imaged in the T cell area of the LN at 18–24 h postinjection. For imaging purposes, we chose areas containing high densities of DCs with dendritic morphology, which were distant from the entry point in the subcapsular sinus. Bone marrow cultures (at day 11) contained >92% CD11c⁺ cells (data not shown). Of these, >97% up-regulated maturation markers upon stimulation with LPS. Injection of BMDCs cultured with OVA plus LPS resulted in proliferation of Ag-specific KJ 1-26⁺ T cells (data not shown).

Adoptive transfer and sample preparation

CD4⁺ T cells with TCR specificity to OVA were isolated by magnetic depletion (Miltenyi Biotec) from the spleens and LN of DO11.10 TCR-transgenic mice (9% regulatory subset). Purified T cells were incubated at 37°C in 2 µM Indo-1 pentaacetoxymethyl ester for 40 min in CO₂-independent medium (Invitrogen Life Technologies) and washed twice. Approximately 8–20 x 10⁶ cells were adoptively transferred by tail vein injection into each recipient mouse 18–24 h after immunization. After allowing 40 min for homing and extravasation of T cells, superficial cervical LN were removed and kept on ice before being placed in the imaging chamber and warmed (37°C) for another 10 min (t = 50 min) before imaging. Little loss or compartmentalization of the Ca²⁺ indicator was evident during the first 5 h after adoptive transfer.

Two-photon imaging

Draining superficial cervical, popliteal, or inguinal LN explants were excised and mounted in a continuous flow chamber bathed in RPMI 1640 medium (BioWhittaker) bubbled with 95% O₂ and 5% CO₂ at 36°C. Multidimensional (x, y, z, time) two-photon microscopy was performed as described previously (32), with modifications for ratiometric imaging of Ca²⁺-dependent fluorescence of Indo-1 (33). Femtosecond-pulsed light at 725 nm was used to excite Indo-1, and the fluorescence emission was monitored at wavelengths of 355–425 and 440–550 nm using a 440-nm dichroic mirror and 390/70 band pass and 550-nm short pass filters (Chroma Technology). The short and long wavelength Indo signals are depicted, respectively, as green and red. Increasing [Ca²⁺] results in increased short wavelength emission and decreased long wavelength emission, resulting in a shift from red to green in pseudocolored images. In some experiments, a third photomultiplier tube collected CMTPX emission (560–650 nm; but depicted blue in images). Imaging volumes (50- to 75-µm depths) with a z-axis resolution of 2.5 µm/step were acquired at 18- to 28-s intervals under control by MetaMorph software (Universal Imaging). Because differential scattering of short as compared with long emission wavelengths at deeper tissue depths may distort ratiometric measurements, we restricted the depths of our imaging volume, standardized the incident laser power, and limited analysis to cells of comparable signal intensity. In pilot experiments, we initially used Fura 2 for calcium measurements, but then switched to using Indo-1, which, unlike Fura 2 permits intrinsic ratiometric imaging by two-photon excitation. The qualitative patterns of calcium signals were similar with both indicators for sham- and OVA-immunized conditions (data not shown). Quantitative calibration of Indo-1 fluorescence, nevertheless, presented considerable technical difficulties due to the greater absorption and scattering of the short excitation and emission wavelengths and the requirement for accurate ratiometric measurements. We thus chose to work on excised LN, rather than intravital node preparations, because the latter exhibit diminished optical quality resulting from superficial fatty tissue and motion artifacts and for other concerns such as anesthesia and surgical trauma intrinsic to the intravital preparation (34).

Image processing and analysis

Photomultiplier settings and digital image adjustments were maintained constant within each experiment to ensure consistent Indo ratio measurements. Endogenous DCs labeled with CFSE were visualized in the long wavelength Indo channel (red) but not in the short-wavelength Indo channel (green). T cell trajectories were tracked to yield centroid coordinates and velocity measurements. Average intensities of short and long wavelength Indo signals were measured at each time point for all T cells that matched acceptable criteria (intensity in both channels at least three times background and not saturated in either channel). The background-corrected ratios of short:long wavelength fluorescence (background-corrected 390:550 short-pass ratio: R) were then used to estimate [Ca²⁺]_i based on in vitro solution calibrations performed individually for each experiment. A Ca²⁺ calibration kit (Molecular Probes) containing zero Ca²⁺ (plus 10 mM EGTA) and 39.8 µM free Ca²⁺ solutions was used to determine the minimal (R_min) and maximal (R_max) fluorescence of 100 µM Indo-1 pentapotassium salt (Molecular Probes), in droplets imaged by the two-photon microscope. [Ca²⁺]_i concentrations were then calibrated using the Grynkiewicz equation (35): [Ca²⁺]_i = K_d*S_f [(R – R_min)/(R_max – R)], where K_d was assumed to be 260 nM, and S_f was the observed emission intensity ratio of the Ca²⁺-free to Ca²⁺-saturated Indo-1 pentaacetoxymethyl ester in the long wavelength channel.

All data are presented as mean ± 1 SEM unless otherwise noted, with statistical significance inferred by the t test. Throughout this manuscript, n denotes the number of instantaneous [Ca²⁺]_i or velocity measurements in the experiment (representative of four experiments from each condition) corresponding to the figure, unless otherwise specified. In some analyses, [Ca²⁺]_i and velocity data from tracked cells were temporally aligned at the onset of an event such as a [Ca²⁺]_i spike, contact with or detachment from a DC. Aligned data points (with 6 events) were averaged at each time point to yield [Ca²⁺]_i and velocity traces that show temporal relationship with an event.

Flow cytometry

BALB/c mice were immunized with OVA plus cyt or cyt only as described above. After 20 h, 5 x 10⁶ CD4⁺ DO11.10 cells were then adoptively transferred into each recipient. At various time points, cell suspensions were prepared by disruption of draining and distal LN. For both cell surface marker and proliferation assays, cells were analyzed by flow cytometry with a gate on KJ1-26⁺ (BD Pharmingen). A CD69 FITC Ab (BD Pharmingen) was used to examine up-regulation of this early activation marker. Proliferation assays used adoptively transferred cells labeled with CFSE. To assess purity and maturity of BMDCs before injection, a portion of the bone marrow culture was set aside and stained with anti-mouse Abs to CD11c, I-A/I-E, and CD86 (eBioscience).

DTH characterization

DTH responses were assayed by immunizing recipient mice in the left ear (25 µl) and scruff (75 µl) with combinations of Ag and cyt. Five x 10⁶ CD4⁺ T cells from DO11.10 mice were injected 20 h after immunization. On day 7 after adoptive transfer, 20 µg of soluble OVA in 10 µl of PBS was injected into the right ear, while the contralateral ear received no injection. Twenty-four hours after Ag rechallenge, both ears were measured and the extent of swelling in the right ear compared to the left ear was used as a measure of the DTH response.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
[Ca²⁺]_i is elevated in DO11.10 T cells after immunization with OVA

We imaged naive T cells inside explanted superficial cervical LN under three conditions: 1) nonimmunized controls (PBS), 2) sham immunization with cyt, and 3) Ag immunization with OVA and cyt (OVA plus cyt). Fig. 1A shows our experimental protocol. CSFE was included in the adjuvant mix, so that sham or Ag immunization of BALB/c recipients resulted in fluorescently labeled DCs migrating from the injection sites to draining LN (25) (see Materials and Methods). One day later (t = 0), naive OVA-specific CD4⁺ T cells that had been loaded with the Ca²⁺ indicator Indo-1 were transferred into recipient mice. After allowing 40 min for homing to lymphoid tissue, draining LN were excised for two-photon imaging. Ratio measurements of Indo fluorescence at emission wavelengths of 355–425 and 425–550 nm were calibrated to derive [Ca²⁺]_i as described in Materials and Methods and are depicted on a pseudocolor scale from red (low Ca²⁺) to green (high Ca²⁺). Fig. 1B illustrates differences in [Ca²⁺]_i between T cells in the nonimmunized (PBS), sham-immunized (cyt), and Ag-immunized (OVA plus cyt) conditions at 2 h after adoptive transfer. The mean [Ca²⁺]_i of T cells from PBS and sham-immunized mice T cells were comparable, whereas [Ca²⁺]_i rose following Ag immunization.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Experimental design. A, Time line of procedures for the three immunization conditions. B, Representative images of Ag-specific T cells in LN cortex of mice in control (nonimmunized, PBS: left), sham-immunized with cyt (middle), and Ag-immunized with OVA and cyt (OVA + cyt: right) conditions. [Ca2+]i, estimated by solution calibration of Indo-1 pentapotassium salt is represented on a pseudocolor scale from red (Ca2+-free) to green (saturating Ca2+). Mean [Ca2+]i measurements from records associated with each snapshot were 118 ± 2 nM (PBS; n = 402), 107 ± 2 nM (cyt; n = 385), and 219 ± 2 nM (OVA + cyt; n = 817).

We first analyzed T cell behavior on a population basis, without regard to specific interactions with identified DCs, so as to identify possible correlations between velocity and [Ca²⁺]_i during basal motility, inflammation, and Ag recognition. [Ca²⁺]_i and velocity measurements were obtained from T cells at successive time points throughout representative imaging records beginning >50 min after adoptive transfer under the three immunization conditions (video S1).⁴ A real-time graphic illustration of [Ca²⁺]_i signaling is provided to illustrate the relationship between changes in Indo-1 fluorescence and the measured Ca²⁺ signal (video S2). In the absence of cognate Ag (PBS and cyt conditions), the distribution of [Ca²⁺]_i measurements centered around 110–120 nM, whereas [Ca²⁺]_i skewed markedly toward higher values after immunization with specific Ag (Fig. 2A). Despite having little effect on [Ca²⁺]_i, inflammation resulting from sham immunization with cyt reduced the peak and average velocities of T cells compared with nonimmunized control (PBS). Ag immunization (OVA plus cyt) decreased velocities even further than with inflammation alone (Fig. 2B). Instantaneous T cell velocity was unrelated to [Ca²⁺]_i in control and inflammatory conditions; but in the presence of cognate Ag (OVA plus cyt) the lowest instantaneous velocities were often associated with striking increases in [Ca²⁺]_i (Fig. 2C). Thus, there are clear distinctions in T cell motility and [Ca²⁺]_i among control (PBS), inflammatory, and Ag-priming conditions. Inflammation reduced basal motility while having little effect on [Ca²⁺]_i, whereas Ag exerted dual effects to reduce motility and increase [Ca²⁺]_i. Together, inflammation and cognate Ag suppress stochastic T cell motility in an additive manner.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Population measurements of T cell motility and [Ca2+]i. A, Distributions of instantaneous [Ca2+]i in nonimmunized (PBS), sham-immunized (cyt), and Ag-immunized (100 µg, OVA + cyt) conditions. Times after adoptive transfer are indicated. Arrows, duplicated in all three panels, mark the mean [Ca2+]i (111 ± 1 nM, n = 347) under sham-immunized conditions for comparison with nonimmunized (118 ± 2 nM, n = 402) and Ag-immunized (143 ± 2 nM, n = 1254) conditions. B, Corresponding distributions of instantaneous T cell velocities. Mean values are: 8.8 ± 0.3 µm/min, n = 389 (PBS); 4.6 ± 0.2 µm/min, n = 361 (cyt); and 2.4 ± 0.1 µm/min, n = 1286 (OVA + cyt). C, Scatter plots, derived from the data in A and B, showing instantaneous [Ca2+]i vs simultaneous measurements of T cell velocity. D, Changes in [Ca2+]i and velocity of T cells in the superficial cervical LN of a mouse immunized with 300 µg of OVA as a function of time (indicated in hours) following adoptive transfer. Panels show scatter plots of instantaneous [Ca2+]i vs velocity, as in C. Crosshairs, duplicated in each panel, indicate the average velocity (6.9 ± 0.2 µm/min, n = 563) and average [Ca2+]i (112 ± 1 nM, n = 576) measured 2 h after adoptive transfer into a sham-immunized mouse. A distinct population of data points at the 50-min time point correlating low [Ca2+]i with low velocities were measured from cells trapped within HEV that happened to be present within the imaging field.

Next, we attempted to determine whether sequential exposure to Ag was necessary before T cells could arrest and undergo robust Ca²⁺ signaling. Fig. 2D shows scatter plots of [Ca²⁺]_i vs T cell velocity at various times after adoptive transfer into immunized (cyt plus OVA) recipient mice. In comparison to control experiments in nodes from sham-immunized mice (marked by crosshairs in all panels of Fig. 2D), little change was apparent in population measurements of velocities or [Ca²⁺]_i at 50 min after adoptive transfer. The response to Ag evolved rapidly within the next 10–40 min (video S3), demonstrated by a large increase in [Ca²⁺]_i and reduction in velocity by 1.5 h, and these changes then persisted for at least another 2.5 h. Thus, early encounters with Ag may be sufficient for maintaining [Ca²⁺]_i signals.

Ca²⁺ signaling modality of individual T cells in LN cortex

We next examined the kinetics of individual T cells to look for specific patterns of Ca²⁺ signals under control, inflammatory, and antigenic conditions. Representative [Ca²⁺]_i traces (blue) and corresponding velocity traces (red) are shown as functions of time for six T cells under each condition (Fig. 3A). The most obvious difference in single-cell Ca²⁺ signals was a markedly greater frequency of [Ca²⁺]_i spike transients under antigenic conditions, as demonstrated by an increase in variance of the [Ca²⁺]_i fluctuations (Fig. 3B). These Ca²⁺ spikes were usually irregular, lacked a clear periodicity, and persisted in T cell populations for many hours after adoptive transfer. Infrequent [Ca²⁺]_i spikes were seen under nonimmunized (PBS) conditions but, surprisingly, their occurrence was even lower under inflammatory (cyt) conditions, as confirmed by a decrease in signal variance (Fig. 3B). [Ca²⁺]_i"trough" levels, calculated as the average of minimum [Ca²⁺]_i values over 5-min intervals throughout each trace, were similar for nonimmunized and sham-immunized conditions (85.6 ± 5.6 nM, n = 32 cells and 92.4 ± 2.5 nM, n = 34 cells, respectively; p = 0.29), but were significantly increased to 125.2 ± 2.5 nM (n = 56 cells, p < 0.001) by immunization with 100 µg OVA.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Ca2+ signals and velocities of representative individual T cells. A, Each pair of traces shows simultaneous measurements of instantaneous [Ca2+]i (blue, top) and instantaneous velocity (red, bottom) for a single T cell. Examples are shown from six cells under control (PBS), sham-immunized (cyt), and Ag-immunized (OVA + cyt) conditions. B, Variance of [Ca2+]i (in arbitrary units) for individual cells under each immunization condition. Red horizontal bars indicate the corresponding means, which were significantly different (p < 0.001) among all three conditions. C, [Ca2+]i spikes under nonimmunized conditions were associated with a slowing in T cell velocity. Traces were derived by aligning [Ca2+]i spikes in 15 cells from two experiments to generate average time-aligned traces of [Ca2+]i (blue, bottom) and corresponding velocity (red, top).

We consistently observed T cells that displayed spontaneous Ca²⁺ spikes, even in the absence of cognate Ag. To determine whether these [Ca²⁺]_i transients were accompanied by changes in T cell motility, we averaged the velocity traces from several cells after time-aligning them to synchronize the rising phase of Ca²⁺ peaks. In the case of basal motility under control (PBS) conditions, this revealed a consistent reduction in velocity beginning roughly coincident with a rise in [Ca²⁺]_i that persisted even after the re-establishment of baseline [Ca²⁺]_i (Fig. 3C). Similar analyses under sham- and Ag-immunized conditions, however, failed to reveal any clear correlation between [Ca²⁺]_i and velocity (data not shown), possibly because motility was already low in both cases.

T cell behavior during Ag-dependent signaling

Previous live-cell imaging studies have reported formation of Ag-specific T cell clusters under priming conditions (24, 25, 29, 32, 36). Soon after T cells were adoptively transferred into Ag-primed mice, we observed the development of robust, dynamic clusters within LN. Clusters were initially sparse in LN at 50 min after adoptive transfer, but their size and density increased consistently with time while the proportion of motile cells decreased. T cells within clusters exhibited Ca²⁺ spiking for the entire duration of the imaging records (>30 min) (Fig. 4A and video S4).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. T cell behavior during Ag-dependent signaling inside the draining LN. A, Snapshot showing several clusters of T cells, as well as isolated cells in the LN of a mouse immunized with 300 µg of OVA. Note the wide variation in [Ca2+]i between cells within clusters. The image is a maximal intensity projection through a 75-µm deep volume, taken from video S4, and was obtained 3 h after adoptive transfer. Scale bar, 20 µm. B, Snapshot of T cells in the same LN as A, taken 1.5 h after adoptive transfer with superimposed cell tracks (18.6-min duration) illustrating representative motilities of clustered (red tracks) and nonmotile/solitary (blue tracks) T cells. The yellow track shows the path of a cell that moved from one cluster to another. Scale bar, 20 µm. See video S5. C, Scatter plot of instantaneous [Ca2+]i and velocity measurements of nonmotile/solitary (blue, left) and clustered T cells (red, right), obtained in a single LN. D, Scatter plot showing average [Ca2+]i vs average velocity for individual T cells from imaging records analyzed in C. Each data point represents mean values for an individual T cell, classified as nonmotile/solitary (blue), and clustered (red). Motile (black) T cells, mostly from early time points, are included for comparison. Mean values for these groups were: nonmotile/solitary, 152 ± 6 nM and 3.0 ± 0.1 µm/min, 22 cells; clustered, 159 ± 4 nM and 3.0 ± 0.2 µm/min; 34 cells, and motile, 113 ± 6 nM and 8.9 ± 0.7 µm/min; 22 cells. Mean [Ca2+]i and velocities were not significantly different (p = 0.34 and 0.78, respectively) between the nonmotile/solitary and clustered groups, but both of these groups were significantly different from motile cells (p < 0.013).

Ca²⁺ signaling and motility of T cells interacting with identified endogenous DCs

The results presented above were obtained without selecting for T cells that were contacting identified DCs. We then proceeded to study Ca²⁺ signaling and motility of T cells while directly visualizing interactions of T cells with endogenously labeled DCs that had been recruited from the immunization site under both sham-immunized (cyt) and Ag-immunized (cyt plus 100 or 300 µg of OVA) conditions.

Measurements of [Ca²⁺]_i in selected populations of T cells that contacted labeled DCs showed dramatic elevations under antigenic conditions as compared with those of sham immunization (Fig. 5A). This effect was also greater than for population measurements of T cells (Fig. 2A) or even T cells within clusters in experiments without visualization of DCs (Fig. 4, C and D). T cells that visibly engaged DCs also had slower velocities after sham immunization as compared with nonimmunized conditions, and their velocities decreased yet further after Ag immunization (Fig. 5B). Elevated [Ca²⁺]_i levels were associated with the lowest velocities for both instantaneous measurements (Fig. 5C) and averaged values of individual cells (Fig. 5D), especially after Ag immunization. The variance of individual [Ca²⁺]_i traces from T cells that contacted Ag-exposed DCs was appreciably greater than that of corresponding sham-immunized experiments (Fig. 5E; p = 1 x 10^–5), indicative of an increase in frequency of Ca²⁺ spiking (Fig. 6). Thus, T cells that were identified as contacting labeled migrant DCs exhibited qualitatively similar, but quantitatively more pronounced signaling and motility characteristics as compared with the overall population of nonselected cells under antigenic conditions.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. [Ca2+]i and motility of T cells that contacted labeled DCs in sham- and Ag-immunized (OVA + cyt) conditions. A, Distributions of instantaneous [Ca2+]i. Mean values were 114 ± 2 nM (cyt; n = 1161) and 225 ± 2 nM (OVA + cyt; n = 1161). B, Corresponding histograms of instantaneous velocities. Mean values were 3.4 ± 0.1 µm/min (cyt; n = 1291) and 2.4 ± 0.1 µm/min (OVA + cyt; n = 1144). C, Scatter plots of instantaneous [Ca2+]i vs velocity; subpopulation in cyt condition with low [Ca2+]i and velocity are attributed to cells trapped within HEV. D, Scatter plot of mean [Ca2+]i vs mean velocity of individual cells pooled from four experiments for sham-immunized (gray) and Ag-immunized (black) conditions. E, Variance of [Ca2+]i traces of individual cells from D under sham-immunized (gray) and Ag-immunized (black) conditions. Bars, mean values.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Instantaneous [Ca2+]i signals and velocities of six representative T cells during contact with endogenously labeled DCs under sham-immunized (A; cyt) and antigenic (B; OVA + cyt) conditions. Each pair of traces shows simultaneous measurements of instantaneous [Ca2+]i (blue; top) and velocity (red; bottom) for a single T cell. Durations of contacts with labeled DCs are indicated by gray bars.

Next, we looked at Ca²⁺ signals of individual T cells during interaction with endogenously labeled DCs. Sham immunization resulted in brief contacts with visualized DCs (20 of 28 contacts had durations <4 min) that were sometimes associated with transient Ca²⁺ peaks (Fig. 6A; right panel of video S6). Overall, however, these Ca²⁺ traces showed low peak amplitudes and baseline trough levels (88 ± 8 nM, n = 12 cells, two experiments) that were little different from that observed in the absence of identified DC contact.

Immunization with OVA promoted more prolonged interactions with identified DCs (20 of 21 contacts had durations >4 min) and stable contacts with DCs often persisted throughout the entire duration of our imaging, regardless of whether the DC was migrating or stationary (video S7). Contacts were associated with frequent, irregular Ca²⁺ spiking, usually superimposed on top of a sustained elevation in basal [Ca²⁺]_i (Fig. 6B). Ca²⁺ spike amplitudes were greater (381 ± 29 nM, n = 23 cells) than in sham-immunized conditions (175 ± 11 nM, n = 24 cells), and the mean trough levels (169 ± 11 nM, n = 12 cells) were significantly greater than the sham Ca²⁺ baseline (88 ± 8 nM, n = 12 cells) (Fig. 6B). Initiation and termination of DC contacts were not obviously associated with any consistent pattern of [Ca²⁺]_i signaling (Fig. 6 and video S8), even when recordings were started soon after adoptive transfer, thereby increasing the likelihood that we captured the early interaction between a T cell and DC.

Ca²⁺ signaling during interactions with exogenous DCs

The experiments described above using endogenously labeled DCs had limitations, in that labeled DCs were sparse (only one or a few per imaging volume) and because T cells may have interacted with Ag-presenting, but unlabeled DCs. We therefore additionally studied Ca²⁺ signals in T cells during interactions with BMDCs that were pulsed with Ag, matured with LPS and fluorescently labeled before s.c. injection (see Materials and Methods). This approach had advantages of increasing the numbers of labeled, Ag-presenting DCs within the LN and of restricting exposure of soluble Ag to the brightly fluorescent, nearly uniform population of mature BMDCs.

Imaging was performed 18–24 h after injection of BMDCs, when both unpulsed (control, LPS) and Ag-pulsed BMDCs (OVA plus LPS) congregated in areas of the T cell zone at high density where they interacted with Indo-labeled T cells. In the absence of Ag, rapidly motile T cells engaged BMDCs serially and maintained low overall [Ca²⁺]_i (left panels, Fig. 7, A and B; video S9). In contrast, a large proportion of T cells showed stable interactions with Ag-pulsed BMDCs as early as 50 min following adoptive transfer, and this proportion increased further with time (right panels, Fig. 7, A and B; video S10). Of 173 cells, 15 (8.7%) were observed to detach from BMDCs after an initial period of contact. During this period of detachment, nine cells (60% of observed detachments) continued to undergo calcium spikes (video S11). In total, we recorded from 66 cells during periods when they were not in visualized contact with BMDC contact, and 35 (53%) of these displayed calcium spikes. Similar to the results with endogenously labeled DCs (Fig. 6), the frequency and amplitude of Ca²⁺ spikes, and Ca²⁺ trough levels all increased in the presence of Ag, whereas cell velocities were slowed (Fig. 7, C and D; cumulative traces not shown). Examination of individual events failed to reveal any clear association between [Ca²⁺]_i and contact with BMDCs for either unpulsed (Fig. 7C) or Ag-pulsed conditions (Fig. 7D). However, after aligning and averaging traces with respect to the initiation and detachment of contacts, a small mean elevation of T cell [Ca²⁺]_i, became evident at the onset of contact with Ag-pulsed BMDCs (Fig. 7E). No comparable change in averaged [Ca²⁺]_i, was evident on detachment from Ag-bearing BMDCs (data not shown) or unpulsed BMDCs, although in the latter case synchronized fluctuations in velocity were evident (Fig. 7F).

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Ca2+ signaling and motility of T cells during interaction with BMDCs. A, Maximum intensity projections showing T cells (Indo loaded, red to green) interacting with homed BMDCs (blue) that were treated with LPS alone (left) or OVA and LPS (right). Images were obtained at times following adoptive transfer of T cells as indicated. The white arrowhead at right (OVA + LPS) marks a T cell cluster. Scale bar, 20 µm. B, Bar graphs show the percentages of T cells displaying different modes of motility and interaction as a function of time following adoptive transfer of T cells in the presence of BMDCs treated with LPS alone (left) or LPS + OVA (right). Three classifications were made: round, nonmotile cells showing stable DC interactions (stable, red), cells with restricted motility that undergo either transient or no visible BMDC interactions (slow, blue), and cells with fast velocity undergoing serial interactions with BMDCs (fast/serial, white). C, Time-lapse sequence illustrating the interactions of individual Indo-loaded T cells with BMDCs (blue) treated with LPS alone (see video S9). Tracks of selected T cells are shown as overlays in the first panels. Elapsed time is indicated at the top right (min:s). Scale bar, 10 µm. The [Ca2+]i and velocity traces of the T cell marked by the white arrowhead are displayed at the right as a function of time, with contacts to one or more BMDCs indicated by gray scale bars. Arrows are color-coded to represent low (red) and high (green) concentrations of [Ca2+]i and correspond to the times indicated in each image snapshot. D, Corresponding image sequence and representative [Ca2+]i and velocity traces for interactions with BMDCs pulsed with OVA and LPS (see video S10). E and F, Averages (more than six cells) of [Ca2+]i (blue) and velocity (red) traces formed after time alignment with respect to the initiation of contact with BMDCs in the OVA + LPS condition (E) and the detachment of T cells from BMDCs in the LPS condition (F). Error bars, SEM. Gray shading shows duration of contact with BMDCs.

	Discussion

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Because our antigenic conditions resulted in near-complete T cell proliferation in the draining LN and establishment of effector function (data not shown), it is likely that these persistent, irregular [Ca²⁺]_i fluctuations contribute importantly to T cell activation. However, we cannot exclude the possibility that initial contacts of newly homed T cells with DCs may have evoked larger [Ca²⁺]_i transients before we were able to begin imaging the node. Despite this, we have found that Ca²⁺ signaling of T cells in lymphoid tissue was strikingly different from many previous in vitro observations. Increased irregularity and unpredictability in signaling is likely due to the dynamic nature of T cell interactions with APC under more physiological concentrations of Ag, in a 3D, highly cellular environment.

Several in vitro systems have been used to study Ca²⁺ signaling during lymphocyte activation, revealing a wide diversity of signaling modes (39); including irregular and periodic oscillations, steady elevations, and abrupt, transient increases (5, 12, 13, 14, 19, 40). Important questions include whether the cell is able to decode all of these different modes and the nature of the activating signal under physiological conditions in the native tissue environment. NFAT, NF-B, and AP-1 are among the most important transcription factor families involved in T cell activation, and all are regulated in a Ca²⁺-dependent manner (41). It has been proposed that gene transcription is activated more efficiently by Ca²⁺ oscillations than by sustained elevations of an equivalent mean level and that specificity among different transcription factors is encoded by the magnitude (18) and frequency of Ca²⁺ elevations (42). For example, human CD4⁺ T cell clones showed Ca²⁺ oscillations and elevated basal [Ca²⁺]_i after encountering Ag presented by APC that were distinguished from Ag-independent Ca²⁺ signals by their increased magnitude and greater spike frequency (7). Our findings are consistent with this signaling pattern in that Ca²⁺ signals under antigenic conditions were distinguished from basal signals primarily by a persistent increase in variance of irregular fluctuations superimposed on modestly elevated plateau levels. In vitro studies showing high amplitude (>800 nM) calcium rises and regularly periodic oscillations may thus be atypical of responses in situ. Ca²⁺ signaling by other immune cells has also been examined in previous in situ imaging studies. In thymocytes, irregular oscillations persisting >2 h, with peaks of 300–1000 nM have been reported to act as a stop signal during positive selection, although APC were not directly observed (33). A recent intravital imaging study showed an immediate increase in [Ca²⁺]_i on contact between MD4 B cells and DCs presenting Ag followed by prolonged arrest (43).

Comparison to in vitro [Ca²⁺]_i measurements in naive CD4⁺ T cells suggests that the [Ca²⁺]_i signals in situ correspond to stimulation by 10–100 pMHC complexes, a level too low to induce Ca²⁺ flux by types of APC other than DCs, and on the steep part of the dose-response relation between Ca²⁺ signaling and number of pMHC (6). Thus, the range of [Ca²⁺]_i signaling in the LN likely represents an optimal compromise between high sensitivity for detection of rare Ags and saturation of Ca²⁺ signaling that may result in tolerance or anergy through activation of NFAT unopposed by AP-1 (44, 45). The oscillatory Ca²⁺ signals observed in vitro must persist for >1 h in order for the T cell to commit to a program of activation (17). Concordant with this, the irregular Ca²⁺ signals we observed in situ persisted throughout the duration of our imaging records (45 min) and probably much longer, suggesting that T cells may integrate Ca²⁺ signals over prolonged periods to distinguish antigenic conditions from the infrequent Ca²⁺ spikes seen in the absence of Ag.

Another difference between in vitro and in situ observations concerns the association between Ca²⁺ signals and contacts with APC. Stimulation of TCR in vitro through Ag presentation results in almost immediate activation of Ca²⁺ signaling, which then subsides rapidly following removal of the stimulus (9, 40). In 3D collagen gel matrices, [Ca²⁺]_i in DO11.10 T cells rose 30–180 s after contact with OVA-pulsed DCs, was maintained while T cells crawled over the DC surface, and fell back to baseline on detachment (11). In contrast, although our in situ observations revealed an average elevation of T cell [Ca²⁺]_i during contact with identified Ag-presenting DCs, individual records generally showed no obvious changes in [Ca²⁺]_i associated with contact or dissociation from DCs. In particular, Ca²⁺ signaling persisted in the absence of identified contact with labeled DCs (e.g., videos S8 and S11). One possibility is that T cells may be exposed to Ag in the LN even in the absence of visualized contact with labeled DCs; e.g., by contact with fine DC dendrites below the limit of our resolution, by transfer of pMHC from DC to T cell (21), by communication within the dense network of resident DCs (46, 47), or by Ag spread among DCs by mechanisms such as uptake of cellular debris or exosomes (1, 48, 49). Alternatively, Ca²⁺ signaling in T cells within the LN may require only sporadic contacts with Ag-bearing DCs to initiate an intrinsic signaling pathway that remains active for an appreciable time even in the absence of Ag (50). Regardless of the specific mechanism, it is clear that Ag conditions lead to irregular Ca²⁺ spikes at higher amplitude and frequency that are clearly distinct from the intermittent basal Ca²⁺ spikes occurring in the absence of Ag. Thus, persistent and irregular Ca²⁺ signaling likely represent the physiological stimulus for T cell activation.

	Acknowledgments

 
We thank Dr. Mark Miller for his participation in early stages of this investigation and Mark Ilagan for help with cell culture and flow cytometry.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants GM-41514 (to M.D.C.) and GM-48071 (to I.P.). S.H.W. was supported by the University of California, Irvine Medical Scientist Training Program and a fellowship from the American Heart Association.

² Address correspondence and reprint requests to Dr. Michael D. Cahalan, University of California Irvine, 275 Irvine Hall, Box 4561, Irvine, CA 92602. E-mail address: mcahalan{at}uci.edu

³ Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; [Ca²⁺]_i, cytosolic Ca²⁺; pMHC, peptide MHC; HEV, high endothelial venule; BMDC, bone marrow-derived DC; DTH, delayed-type hypersensitivity; 3D, three dimensional; cyt, cytokine; Indo-1, 1-[2-amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.

⁴ The online version of this article contains supplemental material.

Received for publication March 12, 2007. Accepted for publication May 22, 2007.

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