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Резюме

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Abstract

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.

Introduction

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 ligands^3,4. 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 activation^5,6,7. 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 c_88-103 (MCC) and ovalbumin_257-264 (OVA), which are presented in the context of the class II MHC I-E^k and the class I MHC H2-K^b, respectively, have been developed (Figure 1). This enables the analysis of both CD4⁺ T cells specific for MCC- I-E^k (expressing the 5C.C7, 2B4, or AND TCRs) and CD8⁺ T cells specific for OVA-H2-K^b (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 remodeling^5,6,7,8,9,10. 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.

протокол

1. Preparation of Stimulatory Glass Surfaces Coat eight-well chambered coverglass with biotinylated poly-L-lysine (Bio-PLL) diluted 1:500 in distilled, deionized water (ddH2O). Incubate for 30 min at room temperature (RT). Wash with H2O. Dry for 2 h at RT. Block Bio-PLL coated surfaces with blocking buffer (HEPES-buffered saline [10 mM HEPES pH 7.4, 150 mM NaCl], with 2% BSA) for 30 min at RT. Dissolve 5 mg of poly-L-lysine hydrobromide in 1 mL of 10 mM NaPO4, pH 8.5. Add 125 μmol (0.55 mg) of NHS-biotin (from a 100 mg/mL stock in DMSO) and check the pH of the reaction. If the pH is below 8.5, add 2 μL of 4 N NaOH to raise it to between pH 8.5 and 9.5. Vortex for 30 min. Quench the reaction with 50 μL of 100 mM glycine dissolved in 20 mM Tris pH 8.0. Spin at full power for 10 min and transfer the supernatant to a new tube. NOTE: Bio-PLL quality is typically compared to previous preparations by coating serial two-fold dilutions of the material onto a 96-well ELISA plate and quantifying biotin content after incubation with alkaline phosphatase coupled streptavidin. Remove blocking buffer. Do not allow wells to dry. Add streptavidin (100 µg/mL in blocking buffer). Incubate for 1 h at 4 °C. Wash in HBS. Fill and invert chamber slide wells 4 – 5 times, removing HBS from the wells. Do not allow wells to dry. Add the biotinylated pMHC ligands and adhesion molecules to the surface. NOTE: MCC and OVA can be photocaged by adding ortho-nitrobenzyl based protecting groups (e.g., nitrophenylethyl (NPE) or nitroveratryloxycarbonyl (NVOC)) to the ε-amino group of key lysines (K12 in MCC, K7 in OVA). Photocaged MCC and OVA can be obtained from commercial vendors. After reconstitution in aqueous buffer, they are refolded into bacterially expressed I-Ek and H2-Kb using established approaches11,12. Validation of photoactivatable MCC or OVA peptide Decage NVOC-protected peptides by UV irradiating with a handheld UV lamp (see Table of Materials) for 20 min at RT. Pulse 1.0 x 105 APCs with 1 µM of nonstimulatory peptide (negative control, e.g. Hb), unirradiated photoactivatable peptide, irradiated photoactivatable peptide, and agonist peptide (positive control, e.g., MCC) for 1 h to overnight at 37 °C. To stimulate T cells expressing the 5C.C7/2B4/AND TCR, use CH12 or CH27 B cells. To stimulate OT1 T cells, use RMA-s or EL4 thymoma cells. Mix the pulsed APCs with an equal number of T cells in 96-well round bottom plates at a final volume of 200 µL. Incubate at 37 °C for 12 – 24 h. Recover the supernatants from each well and analyze IL-2 by ELISA with streptavidin-horseradish peroxidase colorimetric detection. NOTE: Confirmation of the caging status of all peptides by electrospray mass spectrometry prior to refolding into MHC complexes, is strongly recommended. Perform the folding and purification of MHC so as to minimize exposure to light. For example, wrap folding reactions in aluminum foil and carry out gel filtration chromatography with the UV lamp off. NOTE: It has been found that photoactivatable MCC- I-Ek and OVA- H2-Kb induce UV independent T cell activation at high density, possibly due to incomplete functional caging. Hence, the photoactivatable pMHC is diluted into a 10 – 30x excess of nonstimulatory pMHC (e.g. I-Ek containing the hemoglobin64-76 peptide (Hb)) during the immobilization step. This enhances the signal to noise ratio of the subsequent imaging experiment. To photoactivate CD4+ T cells, collectively use a mixture of biotinylated Hb-I-Ek (3 µg/mL), biotinylated photoactivatable MCC- I-Ek (0.1 µg/mL), and biotinylated antibody against the class I MHC H2-Kk (0.5 µg/mL). The anti-H2-Kk antibody encourages 5C.C7/2B4/AND T cells, which express H2-Kk, to spread onto the glass surface without undergoing activation. For CD8+ T cells, use a mixture of biotinylated H2-Db bearing the peptide KAVYDFATL (1 µg/mL), biotinylated photoactivatable OVA-H2-Kb (0.1 µg/mL), and the extracellular domain of the adhesion molecule ICAM-1 (2 µg/mL produced by insect cell culture13). The ICAM-1 encourages close contact formation by engaging the integrin LFA1 on the T cell surface. Apply all protein mixtures in blocking buffer, followed by incubation for 1 h at RT or at least 2 h at 4 °C. NOTE: The molecular density on surfaces of this kind to be ~8000 per µm2 has been previously determined6. Given that photoactivatable pMHC represents ~1/30th of the biotinylated protein on the surface, its density will be ~267 molecules per µm2, prior to decaging. Wash as in step 1.6 and leave in HBS until ready to use. Add 200,000 CD4+ or CD8+ T cells expressing the appropriate TCR into each well and allow cells to adhere at 37 °C for 15 min. Once cells have attached to and spread on the surface, they are ready for photoactivation and imaging. NOTE: Retroviral transduction of effector T cells with fluorescent imaging probes is described in detail elswhere6,7. Calcium signaling can also be studied using untransduced T cells loaded with the calcium sensitive dye Fluo-46. The ratiometric dye Fura-2 is not recommended because it requires excitation in the UV range, which also induces decaging of the photoactivatable pMHC. 2. Image Acquisition Use an inverted TIRF microscope outfitted with a UV compatible 150X objective lens for image acquisition. UV irradiate user-defined regions using a digital diaphragm system attached to a 100 W mercury lamp (HBO). Direct UV light from this lamp onto the sample using a 400 nm long pass mirror. Use image analysis software for localized photoactivation and time-lapse acquisition. In most experiments, monitor probes in both the green and red channels using 488 nm and 561 nm excitation lasers, respectively. Laser light is directed onto the sample using a dual-bandpass dichroic mirror that also transmits in the UV range (to enable decaging). Please see the Table of Materials and Figure 6 for additional information on microscope configuration. After mounting the chamber slide containing the T cells, adjust settings to obtain TIRF or epifluorescence illumination, as necessary. In live mode, select a field of cells that are expressing the fluorescent probe(s) of interest. Establish micron-scale regions for photoactivation beneath individual cells using software control. Begin time-lapse acquisition. Typically, 80 timepoints are acquired, with an interval of 5 s between each time point. This leaves more than enough time for sequential 488 nm and 563 nm exposures, in the case of dual color experiments. After 10 timepoints, photoactivate the selected regions by opening the digital diaphragm shutter for 1 – 1.5 s. After the time lapse is complete, select a new field of cells and repeat the process. 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. Fluorescence intensity Determine the fluorescence intensity (FI) of a background region outside of the cell (FIb). This will be used for background correction. Draw a micron-sized square region outside of the cell and make a mask. To determine the fluorescence intensity, click on Analyze | Mask Statistics. Select Mean Fluorescence Intensity and export the values. NOTE: Procedural details (e.g. 3.1.1.1) refer to Slidebook software (see Table of Materials). Implementation of this protocol using other software packages (e.g., FIJI) will be slightly different. Determine the FI within the photoactivated region for each time point. Select Mask to highlight the region that was photoactivated. To determine the fluorescence intensity, click on Analyze | Mask Statistics. Select Mean Fluorescence Intensity and export the values. Subtract the background FI from the measured FI values within the region. Then, normalize the corrected FI measurements by dividing by the average, background corrected FI of the first 9 frames before photoactivation. ΔF/F = ((FI-FIb)/mean(FI1-9)-FIb)). NOTE: Graph ΔF/F as a function of time. Distance Obtain the x and y coordinates of the center of the photoactivated region. To determine the x and y coordinates of the center of the photoactivated region, select Mask to highlight the region that was photoactivated. Once the region of photoactivation is highlighted, select Analyze | Mask Statistics | Center of Area and export the values. Determine the x and y coordinates for the fluorescent probe of interest for each time point. This is typically achieved through either manual or automated particle tracking. To determine the x and y coordinates of the fluorescent probe of interest, select Manual Particle Tracking and click on the fluorescent probe of interest over time. Once all time points have been tracked, select Analyze | Mask Statistics | Center of Area and export the values. Calculate the distance between the fluorescent protein of interest and the center of the photoactivated region for each time point using the equation: Distance = √((x2-x1)2+(y2-y1)2), in which x2 and y2 are the coordinates of the fluorescent protein of interest and x1 and y1 are the coordinates of the center of the photoactivated region. Graph the distance as a function of time.

Representative Results

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…

Discussion

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 …

Materials

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