Real-time analysis of calcium signals during the early phase of T cell activation using a genetically-encoded calcium biosensor

Abstract

Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux following stimulation with an Antigen Presenting Cell in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naïve T cells in the lymph node occurs in distinct phases. In Phase I, T cells make multiple transient contacts with dendritic cells before entering a Phase II, where they exist in stable clusters with DCs. It has been suggested that T cells signal during transient contacts of Phase I, but this has never been shown directly. Since time dependent loss of calcium dyes from cells hamper long-term imaging of cells in vivo following antigenic stimulation, we generated a knock-in mouse expressing the mCameleon FRET reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro we observed transient, oscillatory, and sustained calcium flux following contact with APC but these behaviors were not affected by the type of APC or antigen quantity, but was however moderately dependent on antigen quality. In vivo, we found that during Phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naïve T cells signal during Phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of Phase II.

Introduction

A hallmark of the adaptive immune response is T cell activation by Antigen Presenting Cells (APCs). T cells are activated when their TCR binds to cognate peptide: MHC complexes on the surface of APCs. The ligation of the TCR initiates a cascade of intracellular signaling events including calcium mobilization, MAP Kinase activity, and activation of the NF-kB pathway. These pathways converge to promote the autocrine secretion of IL-2, which promotes T cell proliferation and differentiation into effector cells, a critical arm of the adaptive immune response. Thus, understanding the regulation of events downstream of the TCR and their functional consequences is of vital importance.

One of the earliest detectable signaling events is a rise in intracellular calcium that occurs within seconds of TCR engagement. This rise of calcium occurs initially from the depletion of calcium stores in the ER, which then stimulates store operated calcium entry from the extracellular space. Increased intracellular calcium activates the phosphatase calcineurin resulting in dephosphorylation of NFAT and its translocation to the nucleus. However, changes in intracellular calcium, can also occur after engagement of other receptors on the surface of the T cell (1). The quality and duration of calcium signals significantly impact proliferation and cytokine production. Notably, artificially-induced calcium oscillations increase NFAT activation and IL-2 production, especially if the overall calcium elevation is low (2). Studies of T cells activated in vitro suggest that oscillations as well as overall intracellular calcium concentrations may control cytokine production in effector T cells (3).

Intravital two-photon microscopy has revealed that events concerning T cell activation in vivo may be more complex (4–6). In vitro, T cells rapidly form stable complexes with APCs and immediately begin exhibiting changes in calcium levels. In vivo, T cells make multiple transient contacts with the APC before arresting their mobility to make long stable contacts (6–8). Von Andrian and colleagues have labeled the transient stage as Phase I and the stable stage as Phase II.

Initially there was controversy about the existence of Phase I since it was seen in some models and not in others. Current evidence indicates that the quantity of antigen is inversely correlated to the length of Phase I (9). One potential explanation is that multiple T cell interactions with the APC are required to generate a productive interaction especially when antigen abundance is low. Alternatively, Von Andrian and colleagues proposed that T cells accumulate signals during multiple transient contact and then make long stable contacts once they have reached a certain threshold of signal (9).

Whether T cells are actively signaling during Phase I interactions is not clear. This would require a sensor that would allow signaling to be detected in vivo by two-photon imaging. Parker and colleagues imaged calcium flux using dye-labeled CD4⁺ T cells to examine the dynamics of early signaling events in the lymph node (10). In their study, they were mainly focused on Phase II interactions and used an antigen dose that exhibited a short Phase I (~50 minutes). Their study clearly shows that the initiation of stable interactions during Phase II is associated with calcium spikes. While this study did not specifically focus on Phase I interactions, they reported cells fluxing calcium after disengagement from the APC (10).

Here we sought to focus specifically on whether signaling occurs during Phase I. We reasoned that if transient contacts between naïve T cells and DCs were generating signals, induced signaling events should be detectable. In contrast, if productive interactions were of low probability and stochastic, no statistically significant signaling would be evident during Phase I interactions. Our strategy entailed monitoring calcium flux as a surrogate for evidence of TCR engagement in vivo. Other studies have used calcium sensitive dyes (10, 11), however, imaging using these methods is limited by time, as these dyes tend to leak out or be actively exported out of the cells. The use of genetically encoded calcium indicators (GECI) circumvents this issue.

While widely used in other fields like neuroscience, only a few studies have used GECIs in T cells. One group introduced a FRET based GECI into activated TCR transgenic cells in vitro, and then imaged calcium responses to antigen after transfer into mice (12, 13). They could detect calcium fluxes as well as oscillations following antigen administration in vivo. Another group studied calcium flux of T Follicular Helper Cells (T_FH) in the germinal center response using GCamP3(14). They found that the magnitude of calcium signaling induced by the T_FH-B Cell interactions in the lymph node was related to antigen quantity. Notably, these previous studies utilizing GECIs analyzed pre-activated or differentiated T cells.

Cameleon is a FRET based sensor that takes advantage of the calcium dependence of calmodulin binding to the M13 peptide (15). Here we generated inducible knock-in mice using a modified form of Cameleon (mCameleon) in which the acceptor fluorophore, YFP was replaced by a brighter variant, Ypet (16). We used these knock-in mice to monitor calcium flux in naïve T cells. We were able to observe distinct calcium patterns in vitro including transient, sustained, and oscillatory as has been previously reported for effector cells. We then showed by peptide titration that the biosensor was sensitive to low concentrations of peptide. Following administration of antigen-loaded DCs, we measured calcium fluxes during Phase I interactions. We found that calcium fluxes were low but increased in the presence of antigen-loaded DCs. Importantly, these fluxes occurred when T cells were not in direct contact with the antigen-loaded DCs. This supports the idea that transient interactions of naïve T cells with DCs induce weak signals that are accumulated over time to initiate Phase II.

Materials and Methods

Mice

All mice were housed under specific pathogen-free conditions in the Washington University animal facilities with the approval of the Washington University Animal Studies Committee. OT-1 Rag1−/− mice were provided by Dr. H. Virgin (Washington University, St. Louis, MO). 5CC7, LLO118, and LLO56 TCR-transgenic mice (17) were provided by Dr. P. Allen (Washington University, St. Louis, MO). Louis, MO). B6.Cg-Tg(CAG-mRFP1)1F1Hadj/J used for purification of CD11c⁺ cells were originally obtained from Jackson Laboratory.

Generation of mCameleon Reporter Mice

The cDNA coding for mCameleon(16) was inserted into the pBS31 targeting vector cells under the control of the CMV minimal promoter containing tetracycline-responsive operator binding sequences (18).The vector, together with the pCAGGS-FLPe-puro vector was used to transfect KH2 embryonic stem cell line (harboring the Rosa26^M2-rtTA allele), as previously described (18). Following electroporation, ES cells were selected with hygromycin and genomic DNA from individual clones was subject to SpeI digestion and Southern Blot using the Col1 3’probe. Laser-assisted injection of selected ES cell clones into 8-cell embryos were performed to generate chimeric mice which were bred for germline transmission of the targeted tetO-mCameleon allele and the Rosa26^M2-rtTA allele.

To induce mCameleon expression in mice, Doxycycline (2g/L; Sigma) was administered in drinking water supplemented with 10g/L of sucrose starting two weeks after birth unless otherwise stated.

Retroviral Transduction of naïve T cells

For retroviral transduction of activated T Cells, viral supernatants were prepared by transfection of Plat-E packaging cells with 30 µg of pMX-mCameleon plasmid using Lipofectamine 2000 (Invitrogen), and viral supernatant was collected 48 hrs and 72 hrs after transfection. 5CC7 T cells were purified from spleens by negative selection using Dynabeads Untouched™ Mouse CD4 Cells Kit (Invitrogen) and purity was checked by flow cytometry. T cells were cultured in IMDM medium supplemented with 10% FBS (Hyclone) in the presence of B10.Br irradiated splenic cells and 5µM peptide specific for TCR-transgenic T cells. After 24hrs and 48hrs of stimulation, retroviral supernatant was added to the T cell cultures and spun for 45 min at 1800 rpm at 25°C in the presence of Lipofectamine 2000 (Invitrogen) and 125 U/ml IL-2. Five days after activation, transduced activated cells were used for in vitro imaging experiments

Generation of Bone Marrow-Derived Macrophages (BMDMs) and Dendritic Cells (BMDCs)

Femurs and tibias from 4–8 C57BL/6J and B10.Br week old mice were manually flushed to harvest bone marrow cells, and red blood cells were lysed in ACK lysis buffer. Cells were cultured in complete DMEM containing 20% of L929 cell-conditioned medium (containing M-CSF) for 8 days to obtain BMDMs. Alternatively, to generate BMDCs, bone marrow cells were cultured in medium containing murine GM-CSF (1000 U/mL) for 8 days. DC and macrophage yield was determined by flow cytometry.

Confocal Microscopy and FRET Analysis

In vitro generated BMDMs or BMDCs were stimulated with IFN-γ (250 U/mL) and loaded with 10µM of the following peptides (unless otherwise stated): wild-type and mutated ovalbumin (OVA) 257–264 (OVAp); listeriolysin (LLO) 190–205 (LLOp); moth cytochrome C (MCC) 88–103 (MCCp); all the peptides were gifts from P. Allen, Washington University. The cells were allowed to adhere overnight to 8-well coverglass chambers (Lab-Tek). Before imaging, wells were washed in Ringers imaging solution (150 mM NaCl, 10 mM glucose, 5 mM HEPES, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2). For naïve T cells, T cells were purified from TCR-transgenic tetO-mCameleon; Rosa26^M2-rtTA mice treated with doxycycline by negative selection using Dynabeads Untouched™ Mouse CD4 Cells Kit (Invitrogen). Cells expressing high levels of Cameleon were sorted with a FACSAria II sorter (BD Biosciences). For activated T cells, after negative selection, the cells were stimulated for 5 days in vitro with irradiated splenocytes (2000 rads) in the presence of 10 µM of specific peptide and 1µM doxycycline, before flow cytometry cell sorting of mCameleon^high cells. Cameleon-expressing T cells were added in Ringers imaging solution right before imaging started. Time-Lapse movies were acquired every 15 seconds by using an Olympus FV1000 confocal microscope in a humidified temperature controlled chamber (37°C). The cells were excited at 440 nm, and DIC images as well as the donor and acceptor emission was detected simultaneously using a 510 nm beamsplitter and 2 photomultipliers with optical filters: 465–495 nm (CFP) and 535–565 nm (YPet). Images were analyzed using Imaris Bitplane Software and MetaMorph (Molecular Devices, Sunnyvale, CA). The YPet/CFP ratio is displayed on a pseudocolor scale, with calculations done on randomly selected cells. To calculate the proportion of cells that were displaying calcium flux, we divided the number of cells that fluxed calcium by the total number of mCameleon-positive cells that were observed in the imaged fields over the course of the experiments.

Proliferation assay

T cells purified from spleen and lymph nodes of doxycycline-treated tetO-mCameleon; Rosa26^M2-rtTA mice were loaded with 5 µM of Cell Trace Violet (Molecular Probes) according to manufacturer’s instructions. T cells were stimulated with anti-mouse CD3 (2C11, 5 µg/mL) and anti-mouse CD28 antibodies (37.51, 2 ug/mL) in the presence of irradiated CD45.1⁺ splenocytes (2000 rads). After 48 hrs and 72hrs, T cells were stained and proliferation was assessed by flow cytometry.

In vivo migration assay

T cells purified from spleen and lymph nodes of C57BL6/J (Ly5.2) were loaded with 5 µM of CMTPX (Molecular Probes) according to manufacturer’s instructions, and mixed at a 1:1 ratio with T cells purified doxycycline-treated tetO-mCameleon; Rosa26^M2-rtTA mice. The cells were injected intravenously into congenic CD45.1+ mice. 24 hrs after transfer, spleen and lymph nodes of recipient mice were harvested and made into single cell suspension and donor T cell migration was assessed by flow cytometry by gating on CD45.2⁺CD3ε⁺ cells.

Flow Cytometry

Single-cell suspensions were generated from the indicated organs followed by Lysis of RBCs with ACK solution. Following Fc Receptor blocking with anti-CD16/32 (2.4G2), cells were stained with the following antibodies obtained from BD Bioscience or Biolegend: anti-mouse CD8-APC-Cy7 (53–6.7), anti-mouse CD8-PECy7 (53.6–7), anti-mouse CD4-PECy7 (RM4–5), anti-mouse CD3-APC (145.2C11), anti-mouse CD3-Pacific Blue (17A2), anti-mouse CD19-PE (6D5); anti-mouse CD45.1-PECy7 (A20); anti-mouse CD45.2-APC (104); anti-mouse CD11c–APC (N418), anti-mouse CD11b–BV421 (M1/70). FACS analyses were performed on a FACS Calibur or a FACS Canto II (BD Biosciences). Data was analyzed with FlowJo software (Treestar).

DC Isolation and Immunization

Dendritic Cells were isolated from spleens of CAG-mRFP1 mice using EasySep™ Mouse CD11c Positive Selection Kit with spleen dissociation medium (Stem Cell Technologies) according to manufacturer’s instructions. Purity was assessed by flow cytometry (>90%). Isolated DCs were loaded with 10 µM OVAp for 2 hours at 37°C. 1×10⁷ DCs were injected with 50 ng of LPS into the footpad of recipient mice.

Two-photon laser scanning microscopy

15 hrs after transfer of OVAp-loaded DCs, wild-type mice were injected with Cameleon expressing OT1+ T cells purified from spleen and lymph nodes. Two to 4 hrs later, excised popliteal or inguinal lymph nodes were placed in a flow chamber and maintained at 37°C by perfusion with RPMI bubbled with a mixture of 95% O₂ and 5% CO₂. Time-lapse imaging was performed with a custom-built two-photon microscope, fitted with two Chameleon Ti:sapphire lasers (Coherent) and an Olympus XLUMPlanFI 20× objective (water immersed; numerical aperture, 0.95) and controlled and acquired with ImageWarp (A&B software). For imaging of Cameleon, the excitation wavelength was 850 nm; Signals from the second harmonic, CFP, YPet and CMTMR were separated by dichroic mirrors (458nm, 510 nm and 560 nm). To create time-lapse sequences, we typically scanned volumes of tissue of 250 × 225 × 50 µm (X,Y,Z; 2,5 µm Z steps) at ~30 second intervals for up to 60 min. Multidimensional rendering and manual cell tracking was done with Imaris (Bitplane) and statistical analysis was performed with GraphPad Prism.

Results

mCameleon is a sensitive calcium biosensor in T cells

In order to measure calcium flux in T cells, we used a modified form of Cameleon (mCameleon), a genetically-encoded FRET-based calcium biosensor. The mCameleon construct that we used is composed of CFP (the FRET donor) and YPet (the FRET acceptor) that are linked by calcium-binding calmodulin and calmodulin-binding peptide M13 (16). In the absence of calcium, the M13 peptide has low affinity for apo-calmodulin, and the excitation of CFP at a 440 nm wavelength does not lead to energy transfer to YPet. The binding of calcium to calmodulin increases its affinity toward the M13 peptide, promoting close approximation of CFP and YPet, resulting in FRET following CFP excitation.

A robust calcium sensor should be able to report physiologically relevant changes in intracellular free calcium concentrations. To assess the viability of mCameleon as a T cell calcium sensor, we determined the sensitivity of mCameleon through in vitro calibration experiments. The plasma membrane was permeabilized and incubated in a range of calcium concentrations. We found that the mCameleon could reliably detect calcium concentrations from 50 nM to 2 µM (data not shown), well within the range of calcium concentrations that occur in the T cell: 100nM-1uM (1). Next, we tested mCameleon sensitivity in T cells contacting antigen-presenting cells (APCs), a key event in T cell priming and activation. We retrovirally transduced T cells from 5CC7 transgenic mice with mCameleon, and imaged them five days later upon restimulation with APCs. We observed that when T cells made contacts with APCs, there was a decrease in CFP emission and an increase in YPet emission when CFP was excited resulting in a change in the FRET ratio (Figure 1A–B).

Figure 1. Calcium signaling in mCameleon-transduced T cells in vitro.

5CC7 TCR transgenic T cells were activated in vitro and transduced with mCameleon. After 5 days, they were added to MCCp-loaded BMDMs, and imaged by confocal. A. DIC and YPet/CFP emission fluorescence ratio of one T cell (arrowheads) interacting with one BMDM (arrows) at different time point after the initial contact. The ratio was color coded from purple for low ratio to red for high ratio. B. YPet/CFP emission fluorescence ratio and YPet (orange line) and CFP (blue line) fluorescence intensity of the T cell shown in A. Arrows indicate the initial contact. C. 30 min after the addition of T cells to BMDMs, EDTA was added to the culture. D. YPet/CFP emission fluorescence ratio of two representative T cells over 6 hours after the addition of T cells to BMDMs.

Interestingly, the YPet/CFP emission ratio often remained elevated for several minutes following the initial increase (Figure 1B). This result could reflect a sustained increase in calcium flux, or more trivially, lack of reversible FRET of the mCameleon reporter. The addition of EDTA to chelate calcium while T cells exhibited elevated YPet/CFP emission ratio immediately decreased FRET (Figure 1C), indicating sustained intracellular calcium. Strikingly, long imaging sessions revealed that elevated YPet/CFP emission ratio could be detected up to 5 hours after the initial increase (Figure 1D). Since calcium dyes leak out of the cell over a period of minutes to hours, the use of mCameleon allowed us to assess calcium signaling at late time-points in vitro for the first time.

mCameleon expression in vivo in doxycycline-inducible mCameleon mice does not affect T cell development, proliferation, or homing

Understanding the molecular events required for T cell priming requires reporters to be introduced into naïve T cells. Retroviral transduction, a common method for genetic manipulation of T cells requires their activation and proliferation. Consequently, this protocol precludes analysis of calcium flux with GECIs in naïve T cells. In order to circumvent this issue, we generated doxycyline-inducible mCameleon knock-in mice. We used KH2 embryonic stem cells which carries a Rosa26^M2-rtTA allele and a modified Col1A1 allele that promotes Flp assisted recombination (18). We targeted a construct containing a tetracycline responsive element upstreatm of the mCameleon cDNA to the Col1A1 locus (referred to as the tetO-mCameleon)

Doxycycline administration of two-week old tetO-mCameleon; Rosa26^M2-rtTA mice induced expression of mCameleon in the spleen and the thymus (Figure 2A–B), as well as in lymph nodes, liver, kidney, pancreatic acini, and kidney tubules (data not shown). Expression was high in all thymocyte subsets (Figure 2C), whereas only ~20–30% of peripheral T cells expressed a high level of mCameleon (Figure 2D). Similarly, high levels of expression were only achieved in a fraction of B cells and myeloid cells. This partial expression is likely due to epigenetic silencing of the collagen I locus that occurs in mature T and B lymphocytes (Wei et al., Immunity 2009). mCameleon expression did not affect T cell development, as T cell numbers were normal in the thymus and the spleen (data not shown). mCameleon-expressing cells also demonstrated normal proliferation in vitro after anti-CD3 and anti-CD28 stimulation (Figure 2E), and normal homing to spleen and lymph nodes after transfer in vivo (Figure 2F). Altogether, these data demonstrate that mCameleon can be expressed in naïve T cells and that its expression does not substantially alter their biology.

Figure 2. mCameleon expression in transgenic mice does not affect T cell development, proliferation, or homing.

A-B. mCameleon expression was assessed by flow cytometry in the thymus (A) and spleen (B) of tetO-mCameleon;Rosa26^M2-rtTA mice treated (black line) or not (grey histograms) with doxycycline (dox) for 3 weeks. C-D. Expression of mCameleon in thymocyte (C) and splenocyte (D) subsets (My = CD11b⁺). Black bars and white bars represent high-expressing cells and low-expressing cells, as gated in B. E. Proliferation of mCameleon-expressing (white bars) or wild-type (black bars) T cells stimulated in vitro with anti-CD3 + anti-CD28 antibodies for 48 hours. F. Equal numbers of CMTPX-loaded wild-type T cells and mCameleon-expressing T cells were co-transferred intravenously to congenic hosts. 24 hours later, the numbers of transferred mCameleon and wild-type T cells recovered in the spleen and peripheral lymph nodes was assessed by flow cytometry.

Calcium patterns are heterogeneous during the activation of naïve T cells

To validate the mCameleon construct, we measured calcium after stimulating naïve T cells in vitro. Diverse calcium signaling patterns have been described previously in pre-activated and polarized T cells (3, 19), however, data regarding naïve T cells is lacking. Therefore, we imaged naïve mCameleon⁺ T cells isolated from TCR-transgenic mice while they interacted with antigen-loaded BMDMs in vitro. We observed three different calcium patterns after the initial calcium increase: 1) a calcium spike followed by a return to baseline in a few minutes (transient), 2) a calcium flux that remains elevated for a long period of time (sustained), or 3) a calcium flux which oscillates for a sustained period of time (Figure 3A). In the sustained pattern, we found that intracellular calcium levels often stayed elevated for several hours (Figure 1D). Interestingly, T cells expressing the same TCR could display all three patterns of calcium flux. To determine whether the calcium signaling patterns could be influenced by the TCR, we bred tetO-mCameleon; Rosa26^M2-rtTA to four different TCR transgenics. We found that the distribution of calcium signaling patterns was specific to the TCR with different TCRs favoring different patterns (Figure 3B). For example, T cells expressing the OT1 and 5C.C7 TCRs displayed mainly sustained calcium patterns, whereas LLO118 and LLO56 TCR-transgenic T cell stimulation displayed all three patterns equivalently. We also found that the T cells exhibiting the sustained pattern had the highest peak ratio compared to transient and oscillatory, however there was no differences in T cell motility at subsequent time points following APC contact (Figure 3C–E).

Figure 3. Heterogeneity of calcium signaling pattern in vitro.

mCameleon expressing TCR-transgenic T cells were purified from spleen and lymph nodes, added to BMDMs that had been loaded with peptide (10µM), and imaged by confocal microscopy. A. YPet/CFP fluorescence emission ratio of T cells displaying transient (top), sustained (middle) or oscillating (bottom) calcium patterns after the initial calcium concentration increase. B. Proportion of T cells displaying transient (white), sustained (grey), or oscillating (black) calcium patterns in different TCR-transgenic populations. C. Intensity of the initial calcium concentration increase, expressed as the YPet/CFP fluorescence emission ratio at the peak of the initial calcium concentration increase divided by the YPet/CFP fluorescence emission ratio before contact. p values of Student t-test are indicated. D-E. Average velocity and arrest coefficient of T cells from 5 to 30 minutes after the initial calcium increase (Not significantly different; Student’s t-test, P>0.05). In C-E, the data from all TCR-transgenic T cells have been pooled.

Calcium patterns are independent of antigen quantity but moderately dependent on antigen quality

Sustained and oscillating calcium patterns have been suggested to influence the efficiency of downstream TCR signaling events (20). Furthermore, it has been demonstrated that stable contacts (synapses) with APCs promote stronger TCR signals in vivo, and that migratory contacts (kinapses) elicit either weak or strong TCR signals (21). Therefore, we tested whether the calcium patterns were related to TCR signal strength and/or the type of contact (synapse or kinapse).

In order to test if sustained calcium patterns were associated with stronger TCR signals, we used three different strategies. To examine the role of antigen dose, we varied the peptide load on APCs. Lower peptide dose diminished the number of T cells displaying calcium flux (Figure 4A), however, amongst the cells that responded, the proportion of cells with sustained calcium patterns, oscillating or transient calcium patterns did not change (Figure 4B–C). This result suggests that the antigen quantity affects the percentage of cells that will respond, but not the magnitude of their response

Figure 4. Calcium patterns are independent of the dose and of antigen-presenting cells, but moderately dependent on quality of antigen.

mCameleon expressing OT-I (A–E) or LLO118 (F–G) TCR-transgenic T cells were added to peptide-loaded BMDMs or BMDCs, and imaged by confocal microscopy. Proportion of total T cells displaying calcium flux (A), T cells displaying transient (white), sustained (grey), or oscillating (black) calcium patterns (B, D, F) and intensity of the initial calcium concentration increase, expressed as in Figure 3C (C, E, G), from pooled data from two to three experiments. A-C. BMDMs were loaded with 10, 1 or 0,1 µM of OVA-peptide (SIINFEKL). D-E. BMDMs were loaded with 10 µM of SIINFEKL (N4), SIIQFEKL (Q4) or SIIVFEKL (V4) peptides. F-G. BMDMs or BMDCs loaded with 10 µM of LLO-peptide were used as antigen-presenting cells. p values of χ² test (A, B, D, F) or Student’s t-test (C, E, G) are indicated if p < 0.05.

Next, we focused on the issue of antigen quality. We utilized the OT-1 system, which has well-established altered-peptide ligands (APLs): N4, Q4, and V4, single residue variants of the SIINFEKL OVA_257–264 peptide. N4 has the strongest affinity and induces the strongest TCR signals, whereas V4 has the weakest affinity and elicits only weak TCR signals. Furthermore, N4 is associated with stable contacts, whereas V4 associates with migratory contacts and Q4 can induce both types of contacts (21). Therefore, if sustained patterns were associated with stronger TCR signaling, we predicted that fewer cells would exhibit sustained signaling when OT1 T cells recognized V4-loaded APCs. We found that the proportion of cells with various calcium patterns was somewhat different between the three peptides, with slightly but significantly fewer cells displaying sustained calcium patterns with the Q4 and V4 peptides compared to the N4 peptide (Figure 4D). Further analysis revealed a modest increase in the peak of calcium flux with N4 peptide compared to Q4 and V4 peptide (Figure 4E), suggesting formation of a more stable contact.

The extent of T cell activation depends not only on peptide: MHC interactions but also on the nature of the APC. In vivo, dendritic cells (DCs) play a pivotal role in the stimulation of naïve T-cells while other APCs such as macrophages and B-cells play a secondary role (22). We hypothesized that DCs might induce more stable calcium patterns than macrophages. However, incubation of LLO118 T cells displayed similar calcium patterns whether they were stimulated with BMDCs or BMDMs (Figure 4F–G). Altogether, antigen quantity or the nature of the APC did not affect calcium patterns that were induced during activation of naïve T cells, whereas antigen quality had a modest impact.

Mature DCs promote T cell calcium flux

Naïve T cells interact with DCs in lymphoid organs in a transient fashion for a period of time that is related to the amount of antigen present on the surface of the DC. At the end of this transient phase (Phase I), naïve T cells make stable contacts with the DC that last for a period of hours (Phase II). An important unresolved issue is whether T cells are signaling during Phase I or whether the increased length of Phase I when antigen is low is due to a requirement for multiple contacts before an antigenic peptide is recognized by the T cell.

To this end, mCameleon⁺ OT-I T cells were transferred into C57BL/6J hosts in the absence of antigen. Explanted lymph nodes were imaged 2–4 hours later by 2-photon microscopy. To measure changes in intracellular calcium with antigen recognition, we added SIINFEKL peptide to the perfusion medium. This induced rapid T cell motility arrest, and a sustained increase in the YPet/CFP emission ratio, that was detectable with doses as low as 100 nM (Figure 5). These results validated the sensitivity of the mCameleon knock-in T cells for calcium imaging studies in vivo.

Figure 5. Sensitivity of mCameleon in vivo.

Lymph nodes of mice that received a transfer of mCameleon-expressing OT-1 TCR-transgenic CD8⁺ T cells were explanted and imaged by 2P. After 11 min of imaging, 10 µM (black), 1 µM (purple) or 100 nM (orange) of OVA-peptide was added to the lymph nodes. Presence of OVA-peptide is indicated by grey shading. A-B. Average velocity (A) and YPet/CFP fluorescence emission ratio (B) of 15 to 30 cells per group. C. Ratio of single cells.

To characterize calcium fluxes in naïve T cells during Phase I interactions with DCs, we purified CD11c⁺ cells from CAG-mRFP1 mice loaded them with SIINFEKL peptide and injected subcutaneously into footpad of WT mice. 15 hours later, mCameleon⁺ OT-I cells were transferred intravenously. Two to four hours later, draining and non-draining lymph nodes were harvested and imaged by 2-photon microscopy. At this time point, few, if any of the T cells had arrested their movement, and most made only short contacts with DCs (average length of contact: 5.7 min) confirming that with this peptide dose and at this time-point, the T cells were in the Phase I period (Figure 6A–B).

Figure 6. Antigen-induced calcium signals in vivo.

mCameleon expressing OT-I TCR-transgenic CD8 T cells were transferred to mice 15 hrs after the injection of Ova-peptide-loaded DCs in the right footpad. Two to four hours later, popliteal lymph nodes were explanted and imaged by 2P. A. Proportion of cells exhibiting slow (<2.5 µm/min), intermediate (2.5 – 6 µm/min) or fast (6 µm/min) average speed in the presence (right popliteal lymph node), or absence (contralateral lymph node) of Ag-loaded DCs. B. Duration of T cell/DC contacts. C-D. YPet/CFP fluorescence emission ratio over time of single cells in lymph nodes without (C) or with (D) DCs. Grey regions indicate contact between T cells and DCs. E–F. Instantaneous velocity (E) and YPet/CFP fluorescence emission ratio (F) of T cells in contact (red) or not (blue) with DCs in the right popliteal lymph node, and in the absence of DC in the contralateral lymph node (black). Each dot represents one cell at one time point. (p<0.0001; ANOVA with post-hoc Tukey’s test) G. Frequency distribution of ratio (color code as in A). Percentages indicate the proportion of T cells with a ratio >1.3. H. Variance of YPet/CFP ratio of T cells in lymph nodes with or without DCs. Each dot represents one cell. p value of Student’s t-test is indicated. I–J. YPet/CFP ratio over instantaneous speed in the absence (I) or presence (J) of DCs (Pearson’s Correlation of linear regression, r²=0.004).

To determine whether there was an effect on T cells when antigen-bearing DCs were present, we measured both cell motility and calcium. Motility measurements showed that the velocity of the T cells when peptide-loaded DCs were present was slower than the velocity of T cells in the absence of transferred DCs (Figure 6E). Imaging of multiple single cells, however, did not demonstrate any cells with a clearly detectable change in FRET ratio when T cells contacted DCs (Figure 6C–D), and this is consistent with previous studies using calcium dye-labelled T cells. Averaging multiple cells, however, demonstrated that intracellular calcium concentrations were clearly elevated in T cells when antigen-loaded DCs were present, compared to T cells in non-draining lymph nodes (Figure 6F). Specifically, a clear and significant proportion of T cells displayed elevated intracellular calcium concentration in draining lymph nodes (Figure 6G). Comparing mCameleon⁺ OT-1 T cells between the draining and non-draining lymph node also showed that the calcium levels had a much broader distribution as compared to the non-draining lymph node where the calcium signal was relatively homogeneous (Fig 6H). Importantly, increased calcium concentration was not dependent on ongoing T cell-DC contacts, as T cells undergoing contact with DCs did not display elevated FRET or decreased velocity compared to T cells that were not in contact with DCs (Figure 6D), and as cells with elevated calcium did not correlate with decreased velocity (Figure 6I–J). These results suggest that during the phase of transient interactions with DCs, T cells flux calcium but at levels that are much lower than occur when stable contacts are formed.

Discussion

While it is clear that TCR ligation results in calcium flux, the exact nature of this phenomenon in vivo remains elusive. What is clear, from a variety of different experimental methods, ranging from studying T cells in suspension to in vivo imaging, is that the nature of calcium flux is context dependent. Thus to understand the nature of T cell activation and the role of calcium signaling it is important to perform experiments in the appropriate physiologic context.

Intravital imaging of T cells loaded with calcium dyes allows for measurement of calcium flux, optimally for 1–2 hours before leakage becomes an issue. This precludes imaging calcium responses that occur later during the adaptive immune response. The use of genetically-encoded calcium indicators (GECIs) that are stably integrated into the genome allows potentially for long-term calcium imaging of T cells. Previous studies used retroviral transduction of activated T cells to express GECIs, precluding its use for naïve T cells (12, 13). We solved this problem by generating a knock-in mouse expressing the mCameleon FRET reporter under the control of a tetracycline responsive promoter.

We found that the expression of the mCameleon reporter did not substantially alter the biology of T cells and was a sensitive indicator of calcium flux at both early and late timepoints. We showed that we could measure calcium fluxes in vitro continuously for up to 5 hours. It had been previously reported that T cells exhibit different patterns of calcium fluxes and it had been suggested that these different patterns might be associated with distinct outcomes for T cell activation. Using a variety of different T cell receptors, we found that the proportion of cells exhibiting a given pattern was dependent on the specific TCR. This bulk behavior of T cells from a specific TCR transgenic was not affected by the type of APC or the quantity of antigen, and was moderately dependent on the quality of the antigen.

Our main goal was to examine the nature of TCR signaling by naïve T cells in lymphoid organs. In vitro, T cells begin signaling within seconds of making contact with peptide-loaded APCs. However, in vivo, the nature of TCR signaling appears to be more complex. The biology of naïve T cells when they enter a lymph node is to first make multiple transient interactions with DCs before making stable contacts. This first transient period, called Phase I, was initially controversial, as some laboratories did not see Phase I interactions. Von Andrian and co-workers demonstrated that the length of the Phase I period is related to antigen dose; using high antigen doses results in a short phase I period that is followed by T cells forming stable, long-lived interactions during the second or Phase II period. Because T cells appear to synchronously enter Phase II, Von Andrian proposed that during the Phase I period, T cells were accumulating signals from transient interactions with antigenic peptide and after a threshold level of signaling was generated, T cells stop moving and Phase II begins. An alternative explanation is that when antigen density is low, T cells required multiple contacts with the DC before a productive interaction with cognated peptide-MHC complex occurs. We hypothesized that if our mCameleon sensor was sensitive enough to detect signaling during Phase I, we could resolve this issue.

We first established that we could detect calcium influx in in mCameleon⁺ OT-1 T cells using 2-photon microscopy in lymph node explants by perfusing cognate peptide into the flow chamber. We observed immediate motility arrest and increased FRET following addition of as little of 100nM peptide indicating that the calcium sensor and that T cells are highly sensitive to low levels of antigenic peptide. Surprisingly, the magnitude of calcium fluxes induced by APC contact was much less after transfer of BMDCs. The analysis of individual cells, did not reveal an obvious increase in calcium during the course of a 30–45 minute imaging period. However, increased calcium was detectable in T cells in the presence of antigen-loaded DCs compared to their absence. This result is consistent with results from Parker and co-workers who found that the calcium signal was much weaker when T cells were stimulated by transferred DCs versus when T cells were stimulated by immunization. One possible explanation is that immunization creates a generalized inflammatory environment in the lymph node and these additional signals can synergize with the TCR to enhance calcium influx.

Parker and co-workers used calcium sensitive dyes to examine T cell signaling in explanted lymph nodes. Most of their studies used immunization with adjuvant and CFSE added to antigen to stimulate and label migratory DCs. Their conditions were relatively strong as they found that naïve T cells were forming clusters and fluxing calcium shortly after they began imaging, about 50 minutes after T cell transfer. This suggests that they were imaging in Phase II. Similarly, when they transferred exogenous antigen-bearing BMDCs for imaging, they saw that most T cells had formed clusters soon after they began imaging.

We established conditions allowing us to image naïve T cells during the Phase I period. During our entire imaging period, T cells were transiently interacting with our labeled, transferred DCs with few, if any T cells forming stable contacts or clusters. In addition, velocity measurements showed that T cells transiently interacting with antigen-bearing DCs were moving with similar velocities to antigen-specific T cells that were not close to any labeled DC. This implies that while the transient interactions stimulate a small but significant increase in calcium, this level of calcium was not sufficient to have an effect on cell motility.

Our data is thus the first to focus on the transient interactions between naïve T cells and DCs during the Phase I period. Our data show that during this period, there is a small but discernable average increase in intracellular calcium but no change in the velocity of the cells. Since this low signal was only seen with antigen-bearing APCs, it suggests that these motile cells have previously had at least one encounter with antigen. The low signal can be explained by the possibility that most of the interactions are non-productive, and thus, no high spikes are seen. The low average signal is due to a previous productive encounter. Importantly, the small but detectable calcium increase in the population of cells suggest that some recognition of antigen had already occurred and that Phase II interactions require at least two or more productive interactions consistent with the model of signal accumulation proposed by Von Andrian.

This suggests that Phase I interactions are a mechanism to allow naïve T cells to assess the density of antigen present on the APC. The requirement for two or more productive interactions within a specific period of time introduces a thresholding phenomenon to activation in vivo. Unlike in vitro, where a single antigenic peptide is sufficient to activate a naïve T cells, this high sensitivity in vivo might lead to poor discrimination between antigenic and non-antigenic peptides, especially if both cognate and non-cognate peptides can contribute to T cell activation.