Evaluation of Controlled T Cell Activation with a Photoactivatable Peptide MHC

1. Preparation of Stimulatory Glass Surfaces

1. Coat eight-well chambered coverglass with biotinylated poly-L-lysine (Bio-PLL) diluted 1:500 in distilled, deionized water (ddH₂O). Incubate for 30 minutes at room temperature (RT).
2. Wash with H₂O.
3. Dry for 2 h at RT.
4. 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.
   1. Dissolve 5 mg of poly-L-lysine hydrobromide in 1 mL of 10 mM NaPO4, pH 8.5.
   2. 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.
   3. Vortex for 30 min.
   4. Quench the reaction with 50 μL of 100 mM glycine dissolved in 20 mM Tris pH 8.0.
   5. 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.
5. Remove the blocking buffer. Do not allow wells to dry. Add streptavidin (100 µg/mL in blocking buffer). Incubate for 1 h at 4 °C.
6. Wash in HBS. Fill and invert chamber slide wells 4 – 5 times, removing HBS from the wells. Do not allow wells to dry.
7. 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-E^k and H2-K^b using established approaches.
8. Validation of photoactivatable MCC or OVA peptide
   1. Decage NVOC-protected peptides by UV irradiating with a handheld UV lamp (see Table of Materials) for 20 min at RT.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
9. 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-E^k and OVA- H2-K^b 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-E^k containing the hemoglobin peptide (Hb)) during the immobilization step. This enhances the signal-to-noise ratio of the subsequent imaging experiment.
10. To photoactivate CD4⁺ T cells, collectively use a mixture of biotinylated Hb-I-Ek (3 µg/mL), biotinylated photoactivatable MCC- I-E^k (0.1 µg/mL), and biotinylated antibody against the class I MHC H2-K^k (0.5 µg/mL). The anti-H2-K^k antibody encourages 5C.C7/2B4/AND T cells, which express H2-K^k, to spread onto the glass surface without undergoing activation.
11. 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 culture). The ICAM-1 encourages close contact formation by engaging the integrin LFA1 on the T cell surface.
12. 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 determined. 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.
13. Wash as in step 1.6 and leave in HBS until ready to use.
14. 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 elsewhere. 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

1. 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.
2. 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 1 for additional information on microscope configuration.
3. 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.
4. Begin time-lapse acquisition. Typically, 80 time points 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.
5. After 10 time points, photoactivate the selected regions by opening the digital diaphragm shutter for 1 – 1.5 s.
6. After the time-lapse is complete, select a new field of cells and repeat the process.