Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

1. Protein Production

1.1. Bilayer-resident proteins: B7-1, ICAM-1, pMHC (e.g., I-E^k/peptide)

1. B7-1 -12H, ICAM-1-12H
   1. Express B7-1-12H and ICAM-1-12H constructs in high quantities as secreted native proteins using a baculovirus expression system.
   2. Purify proteins from culture supernatants via Ni-NTA affinity chromatography, followed by MonoQ anion exchange chromatography and S200 size exclusion chromatography.
   3. Label one aliquot of the purified protein with amine-reactive dyes such as NHS succinimidyl-ester preparations of Alexa Fluor 488 (or FITC), Alexa Fluor 555 (or Cy3), Alexa Fluor 647(or Cy5).
   4. After S200 size exclusion chromatography determine the labeling degree of monomeric proteins according to the dye-manufacturer’s specifications by comparing the protein absorption at 280 nm and the dye absorption at 488 nm (Alexa Fluor 488, FITC), 555 or 552 nm (Alexa Fluor 555 or Cy3) or 647 nm (Alexa Fluor 647 or Cy5).
      Note: This ratio will later be needed to determine the protein density on the SLB from the bulk fluorescence signal of the dye-labeled protein.
   5. Store the unlabeled and fluorophore-labeled proteins at -20°C in PBS plus 50% glycerol.
2. pMHC (here: I-E^k -2x6H or I-E^k-12H)
   1. Refold MHC class II from inclusion bodies expressed in E.coli in the presence of a much cheaper UV-cleavable peptide substitute, which can later be quantitatively exchanged with a fluorophore-conjugated peptide of choice ^5,7.
   2. Purify refolded pMHC complexes by standard techniques (Ni-NTA-affinity chromatography, MonoQ anion-exchange chromatography, S200 gel filtration).
   3. Exchange the UV cleavable peptide with fluorescent peptide ^5,7.
   4. Purify monomeric fluorescent pMHC complexes finally by S200 gel filtration. A representative chromatogram is shown in Figure 3.
   5. Verify quantitative peptide loading by spectrophotometry.
   6. Store proteins at -20°C in PBS/50% glycerol. 

1.2. Generation of single chain antibody fragments (scF_Vs), introduction of cysteines for site-specific labeling

1. Refold scF_Vs from inclusion bodies expressed in E.coli. Here a protocol devised by Tsumoto and colleagues ⁸ is followed in which inclusion bodies are first unfolded in 6 M guanidinium chloride, fully reduced and then refolded in the course of a week by gradually decreasing the concentration of the protein-unfolding guanidinium chloride.
2. Concentrate properly folded scF_V using molecular filter units with a molecular cutoff of 10 KDa.
3. Purify the concentrate by S200 gel filtration.
4. Label monomeric scF_V in the presence of 0.05 mM tris(2-carboxyethyl)phosphine (TCEP) with Alexa Fluor 647 immediately after purification. Perform the labeling reaction best at a molar ratio of protein:dye of no more than 1:2 at RT for no longer than 2 hr.
   Note: TCEP is a phosphorous- based reduction agent and does not react with maleimides at these concentrations ⁹. It can hence be present during the labeling reaction to keep the unpaired sulfhydryl group reduced until it reacts with the dye-maleimide derivative.
5. Purify labeled monomeric scF_V finally via S75 gel filtration. A representative chromatogram is shown in Figure 3.
6. Determine the dye:protein ratio spectrophotometrically.
7. Store proteins at -20°C in PBS/50% glycerol.

2. Calcium Flux Measurements

1. Take a fresh vial containing 50 µg fura-2-AM and dissolve it in 50 µl water-free DMSO.
2. Spin down 10⁶ T-cells in a 5 ml polypropylene round-bottom tube for 2 min at 250-400 g.
3. Resuspend T-cells in 200 µl imaging medium containing Hank's Balanced Salt Solution plus calcium/magnesium and 1% ovalbumin at RT, add 1 µl of the fura-2-AM stock solution (1:200 dilution), mix the cell suspension and incubate at RT for 30 min.
4. Wash the T-cells once in imaging buffer. For this, fill the tube containing the cells with imaging buffer at RT and pellet the cells as described in step 2. Remove the supernatant and resuspend the cell pellet in 200 µl imaging buffer (i.e., at a final cell density of 5 x 10⁶ cells ml^-1). Cells can be used immediately for calcium measurements or stored on ice for up to 3 hr.
5. Place cells on a functionalized SLB in imaging buffer (37° C). As soon as T-cells start contacting the SLB, acquire the following set of images every 15 to 30 sec for 30 min:
   excitation at 340 +/- 5 nm, emission detection at 510 +/- 40 nm
   excitation at 380 +/- 5 nm, emission detection at 510 +/- 40 nm
   DIC (optional)
   Note: Respective exposure times depend on the intensity of the excitation light source. Satisfactory results are obtained when the pixel intensities of 340 nm (380 nm) channel amount to about one fourth (one half) of the maximally possible intensity value. Keep in mind that fura-2 values excited at 340 nm will increase and those excited at 380 nm will decrease upon T-cell activation.
6. 20-25 min into the run, add 50-100 µl of the pre-warmed anti-pMHC blocking antibody at a final concentration of 20-50 µg ml^-1. The antibody saturates all pMHCs and as a consequence T-cells will cease to recognize antigen and stop fluxing calcium. Continue recording images for another 5 – 10 min to acquire the baseline of the intracellular calcium concentration, which corresponds to the non-activated state of the T-cells.
7. Determine the average background fluorescence signal at 340 nm and 380 nm excitation within a region of interest of at least 1,000 pixels, which contains no cell. Background-subtract all measured fura-2 images excited at 340 nm and 380 nm and calculate 340 nm/380 nm intensity ratios of individual cells or a group of cells. Normalize intensity ratios by dividing all ratios through the ratio of the five last time frames (i.e., with complete antibody-blockade of the pMHC).
8. Plot normalized fura-2 ratios against time. Note: When cells and bilayers are in good condition, fura-2 340 nm/380 nm intensity ratios adopt typically values between 2 and 5, 15-45 sec after cells have made contact with the bilayer. Ratios then drop to a value between 1.6 and 2 where they stay constant for at least 20 min or longer. Values should drop to 1 only after the addition of anti-pMHC antibodies. A typical example is shown in Figure 5.

3. Bulk FRET Measurements

3.1. TCR decoration with the H57scF_V

1. Spin down 10⁶ T-cells in a 5 ml polypropylene round-bottom tube for 2 min at 250-400 g.
2. Decant the media, flick the cell pellet gently and add 0.3 µl of scF_V (concentration ~ 1 mg/ml) to the cell suspension. For bulk FRET measurements employ only dye-labeled scFv. For single molecule FRET measurements use a mix of unlabeled scFv (5 to 9 parts, contains no unpaired cysteine) and dye-labeled scFv (1 part, contains one unpaired dye-coupled cysteine).
3. Incubate the cells on ice for 15 min and wash the cells twice through successive centrifugation using ice-cold imaging buffer.
   Note: Cells can be stored on ice without significant loss of bound scF_V (t_1/2 of scF_V dissociation at 0°C ~ 4 hr ²). The purity of primary T-cells derived from TCR-transgenic (and optionally also Rag-1/2 deficient mice) and stimulated in vitro is higher than 98% since T-cells are the only cells proliferating (up to 7 cell divisions) in response to the peptide, which has been added for stimulation to the T-cell culture. B-cells undergo apoptosis and are no longer alive after 7-10 days of cultivation. A few dendritic cells survive yet can be easily discriminated against not only because of their distinct morphology but also because they do not bind the H57 anti-TCR beta scFV fragment.

3.2. FRET measurement via donor recovery after acceptor bleaching

Note: Keep in mind that the half-life of TCR-H57 scF_V complexes amounts to 4 hr on ice, to 50 min at 22.5°C and to 6.8 min at 37°C (and to about 4 hr on ice) ². As long as the H57 scF_V serves as FRET donor, measured FRET yields are not sensitive to H57 scF_V dissociation, however, the signal to noise ratio increases with increased H57 dissociation.

1. Prepare an SLB containing AF647/Cy5-labeled pMHCs as well as unlabeled ICAM-1 and B7 according to Axmann et al. ⁴.
2. Exchange the PBS of the imaging chamber with imaging medium containing Hank's Balanced Salt Solution plus calcium/magnesium and 1% ovalbumin. For this pipet 400 µl of imaging buffer into the well, mix carefully and remove 400 µl from the well. Repeat this procedure 3-4 times. Do not expose the SLB to air at any time.
3. Place the imaging chamber onto the microscope stage and adjust the focus until the fluorescent (AF647, Cy5) SLB comes into clear view.
4. Set up TIRF illumination by translating the focused beam parallel to the optical axis to the periphery of the objective’s focal plane via moving of the periscope’s translational stage.
5. To fine-tune the excitation laser beams in TIRF mode, add T-cells decorated with AF555/Cy3 labeled scF_V and let them settle onto the SLB. Note: Conditions for TIRF illumination are met when the basal cellular membrane is in focus in addition to the SLB and no other parts of the cell come into view when focusing upwards. However, if TIRF is not properly adjusted, parts of the fluorescent T-cell plasma membrane, which are not in contact with the SLB, will appear as a ring. If this is the case, adjust the laser beam with the periscope’s translational stage until TIRF illumination is achieved.
6. Acquire the following six images in the indicated order and in rapid succession (Figure 6):
   (i1, optional) white light, to take an image of the cell,
   (i2, optional) 647 nm excitation (low power) to take an image of the FRET acceptor (pMHC) prior to the bleach pulse,
   (i3) 514 nm excitation (low power) to take an image of the FRET donor (TCR) prior to the bleach pulse,
   (i4) 647 nm excitation (high power) to photo-bleach the FRET acceptor,
   (i5) 514 nm excitation (low power) to take an image of the FRET donor (TCR) following the bleach pulse,
   (i6) 647 nm excitation (low power) to verify complete FRET acceptor bleaching.
7. Keep the time passed between images (i3) and (i5) as short as possible in order to be able to correlate the images for later analysis. Keep FRET donor bleaching at a minimum by employing the excitation at the lowest possible power level, which still allows for proper imaging of the T-cells.
8. Pick a region of interest (ROI), e.g., an entire synapse or an individual TCR microcluster, determine its average intensity in (i3) (= I₍₃₎) and in (i5) (= I₍₅₎). For background subtraction, pick a ROI of the same dimension outside the illumination spot in (3) or (5) and determine its average intensity (I_(background)). To determine the FRET yield perform the following operation:
   (equation 7)
   Note: It should be noted that in relation to the absolute FRET level, it can be necessary to correct for donor bleaching between images (i5) and (i3).

3.3. FRET measurement via sensitized emission

1. Perform spatial donor and acceptor channel alignment with the use of multicolored beads, which fluoresce in all emission channels. The spatial shift between both channels due to chromatic aberration can be determined by super-positioning individual beads and has to be applied for correction of one of the two channels for all following 2-color image-pairs ¹⁰.
2. Determine the degree of donor bleedthrough with the use of an SLB containing the FRET donor fluorophore alone. It is also feasible to use FRET-donor-labeled T-cells on a lipid bilayer with unlabeled pMHC. Background is first determined outside the illuminated field of view and then subtracted from both channels. This way the average background-corrected intensities of two corresponding ROIs (I _{donor channel} and I _{acceptor channel}) are determined. Calculate the bleedthrough coefficient (BTC) as follows:
   (equation 8)
   Note: This BTC is a constant for a certain dye and filter set-combination.
3. Calculate the FRET donor bleedthrough image as follows:
   (equation 9)
4. Determine acceptor cross-excitation by exciting an SLB containing the FRET acceptor fluorophore alone (e.g., I-E^k/MCC-Alexa Fluor 647) first with FRET donor excitation light (e.g., 514 nm) and then with acceptor excitation light (e.g., 647 nm). Use background-subtracted images within the FRET acceptor channel to calculate the cross-excitation coefficient (CEC) as follows:
   (equation 10)
5. As the CEC depends on the laser intensity used for donor excitation, determine it on each measurement day. Use the resulting CEC to calculate the image solely generated through cross-excitation.
   (equation 11)
6. Calculate the FRET image corrected for bleedthrough and cross-excitation as follows:
   (equation 12)
   Note: Absolute FRET signal (but not the relative FRET yield) is sensitive to the dissociation of the H57 scF_V from T-cell membrane bound TCR. To avoid excessive loss of the TCR-FRET probe, aim to perform measurements at 37°C within the first 2 min after the addition of the T-cells to the bilayer. Measurements with quantitative (≥ 95%) TCR labeling are only possible at or below 22.5°C within the first 3 min after addition of T-cells to the bilayer.

4. Single Molecule FRET Measurements

1. Adjust the power of both lasers to give rise to an intensity of 1-5 kW/µm² at the specimen. For more information please refer to Axmann et al. ⁴.
2. Label T-cells as outlined above with a mix of unlabeled scF_V (5-9 parts) and Cy3/AF555-labeled scF_V (1 part). Note: This way only a fraction of TCRs is marked with the FRET-donor. This reduces the number of detectable interactions, but noise generated from donor bleedthrough is also decreased significantly (by about the 5^1/2 to 10^1/2), which is critical when resolving individual single molecule FRET events.
   Note: The dissociation of the H57 scFV probe from the TCR does not affect the measurements, as it occurs in a much larger time range (minutes to hours) than the dissociation of the TCR from SLB-bound pMHC (sub-second to second range).
3. Place an SLB featuring AF647-labeled pMHCs as well as ICAM and B7 on the microscope stage and adjust the focus so that the bilayer comes into clear view.
4. Optional: Insert a slit aperture into the excitation pathway (as shown in Axmann et al. ⁴) to mask the majority of the field of illumination except the synapse. This way unbleached I-E^k/MCC (C)-Alexa Fluor 647 FRET acceptor molecules can move into the area of illumination.
5. Add H57 scF_V decorated T-cells to the bilayer (with imaging buffer), and wait until synapses appear in the field of view.
6. Take in rapid succession a sequence of 10 to 20 image sets using 2-color detection:
   i1) excitation 514 nm
   i2) excitation 647 nm
7. Expose images for 1 to 5 msec and acquire as a set of images.
8. To assess the identity of single molecule FRET events, apply the same correction regarding donor bleedthrough and acceptor cross-excitation. Moreover, single molecule FRET events have to align with single acceptor molecules and should appear and disappear in one step (see also Figure 7).
9. Off-rate determination
   1. Record traces of single molecule FRET events for several acquisition time frames.
      Note: In this example (in italics) the off-rate between the 5c.c7 TCR and I-E^k/K3 is measured at 25 °C with four different delay times (42 msec, 490 msec, 1,007 msec, 1,989 msec).
   2. List FRET traces according to their trace lengths as shown in table 1.
   3. Convert table 1 into an inverse cumulative decay function (table 2) as shown (colored numbers are taken from table 1).
   4. To normalize the resulting decay function, divide the number of traces of table 2 by the sum of all traces in that particular group. Plot the normalized values against the number of time frames. When omitting the last time frame, which contains a zero, the decays can be readily fitted with a single exponential function (Figure 8A).
   5. As shown in Figure 8B, plot the expectation value <n(t_lag)> , i.e., the negative inverse of the exponent of the decay function determined above against the delay time t_lag employed (in this example: x= t_lag = 0.042 s and y= <n(t_lag)> = 1/0.662 = 1.511, x = 0.49 s and y = 1/0.902= 1.101, x = 1.007 s and y = 1/1.131 = 0.884, x = 1.989 s and y = 1/1.591= 0.629).
   6. Fit τ_off and <n_bleach> based on equation 3. This can be done using a non-linear fitting function of a scientific data analysis program such as Origin.
      Note: The best fit shown in this example yields a τ_off of 2.12 +/- 0.23 s and a <n_bleach> of 1.53 +/- 0.06 sec.
   7. Calculate the half life of the interaction t_{1/2 off} with t_{1/2 off} = τ_off • ln(2) (in this example: 1.47 s).
10. 2D-K_D determination
    1. Determine the conversion factor C for the FRET dye pair employed with the use of equation 6 and as outlined above (Figure 4A).
    2. Determine the FRET yields for individual TCR microclusters or entire synapses (Figures 3B and 5).
    3. Using equation 4 convert all individual FRET yields into TCR occupancies (Figure 4C).
    4. Apply equation 11 to convert all TCR occupancies into 2D-K_Ds. As is indicated below, synaptic binding is inhomogeneous. A meaningful measure for synaptic K_Ds is the median (indicated in red) of all measured microclusters (Figure 4D).
11. Calculation of 2D-k_ons
    1. Calculate k_on with the law of mass action with k_on = k_off/K_D and the experimentally determined k_off and K_D values.
       Note: The synaptic k_off for the experiment shown in Figure 4 (I-E^k/MCC interacting with 5c.c7 TCR at 25 °C) is 0.41 s^-1. Hence the K_D plot (Figure 4D) can be converted into a k_on plot as shown in Figure 4E.