This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.
T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.
T lymphocytes (T cells) play a central role in cellular immunity by recognizing antigenic peptides displayed in the context of cell surface MHC. Antigen recognition, which is mediated by the TCR, drives the differentiation of naïve T cells and promotes the delivery of cytolytic and communicative responses by effector populations. TCR engagement also induces dramatic changes in cellular architecture. Within minutes, the T cells gloms onto the side of the antigen-presenting cell (APC), forming a polarized interface known as the immunological synapse (IS)1,2. The IS potentiates T cell effector responses by enabling the directional release of cytokines or, in the case of cytotoxic T lymphocytes (CTLs), lytic proteins that destroy the APC.
TCR engagement by pMHC induces the rapid phosphorylation of multiple downstream adaptor molecules, including Linker for the Activation of T cells (LAT), which ultimately promotes robust remodeling of the synaptic cytoskeleton2. Cortical filamentous actin (F-actin) drives T cell spreading over the APC surface, and then resolves into an annular structure characterized by F-actin accumulation at the IS periphery and depletion from the center. F-actin ring formation is tightly coupled to the reorientation of the microtubule organizing center (MTOC, also called the centrosome in T cells) to a position just beneath the center of the interface. Both events occur within minutes of initial antigen recognition and establish the architectural context in which subsequent activation events and effector responses occur.
To study IS formation, various labs have developed approaches in which the APC is replaced by a glass surface that either contains immobilized TCR ligands or supports a lipid bilayer that itself contains the ligands3,4. T cells form IS-like contacts on these surfaces that can be imaged by total internal reflection fluorescence microscope (TIRF) or confocal microscopy, enabling high-resolution studies of early T cell activation and IS formation.
Although these approaches have allowed for excellent visualization of the fully assembled IS, much of the signaling following TCR:pMHC ligation occurs within seconds, complicating efforts to determine the sequence of events following TCR activation accurately. To circumvent this issue, a photoactivation approach has been developed, in which photoactivatable pMHC is used to achieve spatiotemporal control of TCR activation5,6,7. In this system, T cells are attached to glass surfaces containing photoactivatable pMHC that is non-stimulatory to the TCR until irradiated with ultraviolet (UV) light. UV irradiation of a micron sized region of the surface beneath the T cell removes the photocage creating a stimulatory zone that can be recognized by the T cell. Subsequent signaling events and cytoskeletal remodeling are then monitored using genetically encoded fluorescent reporters. Two photoactivatable versions of antigenic peptides, moth cytochrome c88-103 (MCC) and ovalbumin257-264 (OVA), which are presented in the context of the class II MHC I-Ek and the class I MHC H2-Kb, respectively, have been developed (Figure 1). This enables the analysis of both CD4+ T cells specific for MCC- I-Ek (expressing the 5C.C7, 2B4, or AND TCRs) and CD8+ T cells specific for OVA-H2-Kb (expressing the OT1 TCR).
Over the past decade, the TCR photoactivation and imaging approach has been utilized to establish the precise kinetics of early TCR signaling steps and also to identify the molecular pathways governing polarized cytoskeletal remodeling5,6,7,8,9,10. For example, the assay was instrumental in determining that centrosome reorientation toward the APC is mediated by a localized gradient of the lipid second messenger diacylglycerol centered at the IS. It is anticipated that this methodology will continue to be valuable for applications that demand high-resolution imaging analysis of T cell function.
1. Preparation of Stimulatory Glass Surfaces
2. Image Acquisition
3. Data Analysis
NOTE: Localized photoactivation of immobilized ligands creates a well-defined, stationary stimulatory region that can be used for quantitative analysis of signaling responses and cytoskeletal remodeling events. Analysis protocols typically involve either quantifying fluorescence intensity within the irradiated region or using the irradiated region as a positional endpoint for distance measurements (e.g. for assessing polarization of the centrosome to the irradiated region). Both analysis protocols are described below. Various interactive image analysis programs can be used to make intensity and distance measurements, which can then be output for additional processing and analysis.
The photoactivation and imaging approach allows for observation and facile quantification of rapid, polarized signaling responses. To illustrate its capabilities, reproduced here is an experiment examining the spatiotemporal correlation between TCR-induced DAG accumulation and centrosome reorientation. 5C.C7 T cell blasts were retrovirally transduced with two fluorescent reporters: a DAG biosensor containing the tandem C1 domains from protein kinase C-θ linked to GFP (C1-GFP) and RFP-tubulin to monitor the centrosome. The T cells were then attached to chambered coverglass containing photoactivatable MCC- I-Ek. C1-GFP was imaged using TIRF microscopy and RFP-tubulin in epifluorescence mode. As shown in Figure 2, localized photoactivation of the surface beneath the T cell induces the accumulation of DAG in the irradiated zone. This is followed within seconds by the reorientation of the centrosome to the same region.
C1-GFP accumulation can be quantified by calculating the normalized fluorescence intensity, after background correction, at the center of the photoactivated region over time (Figure 3). Centrosome reorientation in response to photoactivation can be quantified by calculating the distance between the centrosome and the center of the photoactivated region as a function of time (Figure 4).
This method can be used to examine a wide array of rapid, polarized signaling responses, essentially anything that can be monitored using a fluorescent probe. For example, TCR photoactivation is utilized to monitor early signaling microcluster formation using fluorescently labeled Grb2 (Grb2-GFP) (Figure 5). Similar to the DAG accumulation and centrosome reorientation responses, Grb2-GFP polarization occurs shortly following photoactivation (Figure 5), and can be quantified using the normalized fluorescence intensity approach.
Figure 1: Photoactivation system using photocaged MCC peptide.
(A) Photocaged strategy for MCC peptide. A NPE group is attached to the lysine residue of the MCC peptide. UV irradiation results in the removal of NPE, restoring MCC to its native form. (B) When NPE is bound to MCC, the 5C.C7 TCR cannot bind to MCC. Removal of NPE results in recognition of the 5C.C7 TCR to the native MCC peptide. (C) The photocaged strategy has been adapted to work with CD8+ T cells, in which photocaged OVA peptide is used to activate the OT-1 TCR upon UV irradiation.
Figure 2: Time-lapse montage of DAG accumulation and centrosome reorientation following photoactivation of TCR in 5C.C7 T cell.
5C.C7 cell expressing a DAG biosensor, containing tandem C1-domains of PKC- fused to GFP (C1-GFP) and Tag-RFP Tubulin (RFP-Tub), a fluorescent reporter for the centrosome. The montages illustrate the polarized response of 5C.C7 T cells over time. The yellow oval and text indicate the time and location of photoactivation. Over time, C1-GFP accumulates at the photoactivated region, followed by centrosome reorientation towards the photoactivated region. Scale bar: 10 µm.
Figure 3: Quantification of DAG accumulation at the photoactivated region
DAG, C1-GFP, accumulation at the photoactivated region (left) can be quantified by calculating the normalized fluorescence intensity, after background correction, at the photoactivated region (yellow circle with blue shading) over time. Data are normalized relative to the nine frames obtained just before photoactivation. C1-GFP enrichment at the photoactivated region can be calculated by the division of the mean fluorescence intensity in the irradiated region by the mean fluorescence intensity of the entire cell, following background correction. The equation: ΔF/F = ((FI-FIb)/mean(FI1-9)-FIb)), in which FI is the mean fluorescence intensity, can be used to calculate the normalized fluorescence intensity. The normalized fluorescence intensity at the photoactivated region can then be graphed as a function of time (right). Purple line indicates the time of photoactivation. Scale bar: 10 µm.
Figure 4: Quantification of centrosome reorientation.
Determine the coordinates of the centrosome (RFP-Tub) over time by manually tracking the centrosome path toward the photoactivated region (white arrow) at each time point (left). Determine the coordinates of the center of the photoactivated region (yellow circle). The distance between the centrosome and the center of the photoactivated region can be calculated at each time point using the equation for distance = √((x2-x1)2+(y2-y1)2), in which x2 and y2 are the x and y coordinates for the center of the area of the centrosome, and x1 and y1 are the x and y coordinates of the center of the photoactivated region. The distance of the centrosome from the center of the photoactivated region at each time point can then be graphed as a function of time (right). Purple line indicates the time of photoactivation. Scale bar: 10 µm.
Figure 5: Grb2-GFP microcluster formation.
(A) Representative 5C.C7 cell expressing fluorescently labeled Grb2 (Grb2-GFP). The montages illustrate the polarized response of Grb2-GFP overtime. The yellow oval and text indicate the time and location of photoactivation. Over time, Grb2-GFP accumulates at the photoactivated region. (B) Quantification of Grb2-GFP fluorescence intensity at the photoactivated region over time. N = 15 cells. (C) Quantification of Grb2-GFP fluorescence intensity in a control region outside of the photoactivated zone. N = 15 cells.
Figure 6: Microscope configuration for photoactivation and TIRF imaging.
488 nm and 561 nm lasers are used for TIRF and epifluorescence imaging of green and red probes, respectively. UV light for photoactivation is taken from a mercury (Hg) lamp attached to a digital diaphragm system capable of illuminating small, user-defined regions. Light from the Hg lamp/diaphragm is reflected onto the sample by a longpass mirror positioned beneath the dichroic mirror that reflects the imaging lasers. The dichroic mirror must be designed to pass UV light. Please click here to view a larger version of this figure.
In recent years, light has emerged as an excellent tool for spatiotemporally controlled activation of cellular processes. Various methodologies have been developed, each with associated advantages and disadvantages. The system described here, which is based on the decaging of immobilized, extracellular ligands, is ideally suited for the analysis of rapid, subcellular, polarized signaling responses. This approach has been applied to examine IS formation in T cells as described above. Additionally, caged ligands for other receptors have been engineered, enabling us to apply the same strategy to assess inhibitory signaling in natural killing cells and nuclear translocation of the transcription factor NF-κB in response to toll like receptor signaling14,15.
In this protocol, stimulatory ligands are immobilized to ensure that they remain as a stationary, polarized source of stimulation for the duration of the imaging experiment. This approach is well suited for studies of cell polarization. It should be noted however, that the TCR photoactivation approach is easily adapted for temporally controlled T cell activation on supported lipid bilayers. Obviously, one cannot achieve sustained, polarized stimulation in this context. This may be unnecessary, however, given the experimental objectives.
The system is also quite flexible with respect to the UV light source for decaging. Currently, a 100 W Hg lamp is used for focused illumination, however a pulsed nitrogen laser (pulse width 4 ns, pulse energy 120 mJ), was previously used6. At times, a handheld UV lamp (365 nm, 4 W) has been positioned over the imaging sample in order to decage larger regions of photostimulatable ligand. The specific features of the decaging system will in each case depend on the technical requirements of the experiment in question.
When implementing this system, it is important to confirm that the observed responses are specific for the photoactivation event, and not the result of spurious receptor activation or UV-induced photodamage. To rule out these artifacts, two specificity controls can be performed. First, fluorescence intensity in cells that are not directly irradiated during photoactivation experiments can be monitored. If the surface is properly caged, appreciable responses in these cells should not be observed (for example, Figure 5C). Second, experiments in which T cells are UV-irradiated on surfaces lacking photoactivatable ligands can be performed. This control is critical for identifying UV-induced effects that are independent of photoactivatable ligand (for example, see reference 6). During system optimization, it is also extremely helpful to have at hand a robust cellular imaging response (e.g., Grb2-GFP recruitment) that is both fast and localized. Assessing correlations between signaling events and photoactivation is much more straightforward if those events are spatiotemporally proximal to receptor stimulation.
Although the photoactivation approach offers exquisite spatiotemporal control over receptor signaling, it lacks the adaptability afforded by transfectable optogenetic systems16,17,18,19,20,21,22. Certain optogenetic proteins are also photoreversible, providing conditional control that is not available with the photoactivation approach described here. In optogenetic systems, however, constraining the diffusion of photoactivated proteins is technically challenging. This complicates the analysis of polarized signaling, particularly in a subcellular context.
One should also note that the engaging entity of this photoactivation system is a glass surface, which cannot recapitulate all aspects of a bona fide cell-cell interaction. To circumvent this issue while maintaining spatiotemporal control of signal initiation, investigators have used an optical trap system to bring an APC into contact with a T cell, allowing for the onset of cytoskeletal rearrangements and IS maturation to be visualized in real time23. Although this system enables the precise initiation and visualization of cytoskeletal dynamics using an actual APC, it is relatively low throughput and is not compatible with TIRF illumination, which adversely affects image quality.
In conclusion, the method described in this protocol provides spatiotemporal control of TCR-induced signaling within the T cell, ultimately allowing for the elucidation of the fast biochemical changes following TCR activation. This system provides us with a means to better understand how the signaling events following TCR activation promote T cell polarization, IS formation, and ultimately the delivery of effector responses.
The authors have nothing to disclose.
We thank members of the Huse lab for advice and assistance. Supported by the US National Institutes of Health (R01-AI087644 to M.H. and P30-CA008748 to Memorial Sloan-Kettering Cancer Center).
Nunc Lab-Tek Chambered Coverglass | Thermofischer Scientific | 155361 | |
Poly-L-lysine hydrobromide | Sigma-Aldrich | P2636 | Will need to make Biotinylated Poly-L-Lysine |
EZ-Link NHS-Biotin | Thermofischer Scientific | 20217 | Will need to make Biotinylated Poly-L-Lysine |
Streptavidin | Thermofischer Scientific | 434301 | |
BirA-500: BirA biotin-protein ligase standard reaction kit | Avidity | BirA500 | Will be used to biotinylate proteins |
Biotinylated Hb I-EK | For protein folding, see reference 6. For biotinylation, use BirA kit | ||
Biotinylated NPE-MCC I-EK | Anaspec | Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec | |
Biotinylated αH2-Kk antibody | BD Biosciences | 553591 | |
Biotinylated NPE-OVA H2-Kb | Anaspec | Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec | |
Biotinylated KAVY H2-Db | Anaspec | Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec | |
Biotinylated ICAM1 | For protein folding, see reference in protocol. For biotinylation, use BirA kit | ||
Hand held UV lamp | UVP | UVGL-25 | Lamp is held < 1 cm from the sample. 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging. |
Olympus IX-81 OMAC TIRF system. | Olympus | Additional information about the imaging system can be found in Figure 6 | |
Mosaic digital diaphragm | Andor | ||
Slidebook software | Intelligent Imaging Innovations |