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

In this video, we will show how to measure the kinetics between the T-cell antigen receptor and peptide loaded major histocompatibility complex or PMHC in C two that is within the immunological synapse between a T-cell and a planar supported lipid blay. We will briefly guide you through important aspects of protein production and verifying the functional integrity of the lipid blay via calcium flux measurements. For more detailed information on how to prepare lipid blay and how to set up a single molecule fluorescence microscope, please refer to our parallel contribution In the Journal of Visualized Experiments, we will then explain in detail how to conduct and analyze first or resonance energy transfer measurements in bug and on a single molecule level.

To deduce the kinetics of synaptic T-cell receptor PMHC binding to explain our fret based strategy used in our overall approach, We start out with the crystal structure of the T-cell receptor or TCR here in blue as it binds its antigen, the peptide loaded MHC molecule or PMHC shown in yellow and red. We also introduce a single chain fragment of the variable domains from a T-cell receptor beta reactive antibody termed age 57. For quantitative fre based measurements, it is mandatory to label both the fre donor, in our case, the single chain fragment and the fre receptor, the PMHC in a site specific manner.

This is feasible for recombinant proteins through the introduction of an unaired cysteine, which can then be covalently bound to mite conjugated Fluor force. As shown, we attach the fed donor Alexa Fluor 5 5 5 or side three to the single chain fragment and the fret acceptor. Alexa flow 6, 4 7 or SI five to the C terminus of the MHC bound peptide.

The Indy distance as shown in this composite structure is about 41 angstrum, even less than the first radius, the distance of half maximal energy transfer between the two dyes. In our experiments, we employ planar glass supported lipid bilar as surrogate antigen presenting cells. These are functionalized with PMXC, the atory molecule B seven one and the adhesion molecule I chem one.

This way imaging can be performed in total internal reflection fluorescence mode, which illuminates Fluor force in close proximity to the interface, thereby reducing cellular background substantially. Fret is only detectable when single chain fragment labeled TCRs are engaging by layer embedded PM hcs. This allows us to visualize T-C-R-P-M-H-C binding And then calculate the binding kinetics in C two properly folded histo gene Tact.

Extracellular portions of P seven one and I chem one are purified from supernatants of insect cell cultures infected with the corresponding BUC virus. Purification is performed in three steps, starting with nickel anti aros affinity chromatography, followed by an iron exchange and size exclusion chromatography. Histo tag MHC Class two heterodimers are expressed in e coli as inclusion bodies and refolded in vitro at four degrees Celsius and the presence of a space holder peptide through rapid dilution in redox buffer.

The space holder peptide will later be cleaved via UV light exposure. Properly folded PMHC complexes other then purified and concentrated the space holder. Peptide is cleaved and substituted at pH 5.2 with the fluorescent peptide.

The final product is stand size purified. The single chain fragment is expressed with an unaired cysteine as inclusion bodies in e coli refolded in vitro through stepwise dialysis and size purified. It is then labeled via its unaired cysteine with a maide conjugated dye and size purified.Again.

All recombinant proteins are finally dialyzed against PBS containing 50%glycerol concentrated to one milligram protein per milliliter and start long term at Minus 20 degrees Celsius. Use the ratio metric URA two dye To measure intracellular calcium levels in T cells as a means to verify the stimulatory potency of the lipid blay. Load the T-cell blast with the URA 2:00 AM ester.

This compound enters the cells via its AM moty, but it's then trapped within through the action of esterases, which cleave off the AM part. Add fira 2:00 AM to about 3 million T cells at a final concentration of five microm MO per liter. Transfer the cells from the 24 well plate to a new tube and incubate at room temperature for 30 minutes.

Wash the cells once in imaging buffer containing Hanks buffered saline solution including calcium and magnesium and 1%of albumin. To monitor intracellular calcium, use an automated UV capable inverted microscope system. Start the timing protocol and add fewer two loaded T cells to the functionalized lipid bilayer placed in focus on the microscope stage, acquire in regular time intervals, A DIC image and two fluorescent images excited with 340 and 380 nanometer UV light.

Immediately after making bilayer contact T cells adopt elevated calcium levels as read out by the URA two ratio 15 minutes into the run. Add a blocking antibody to the cells which saturates all PMHC antigens. As a consequence, intracellular calcium levels drop down to those of the nonactivated state, which serve later as a reference.

As soon as the blocking antibody present levels decrease to a value, which reflects the nonactivated state, Which we normalize to one for fret measurements, T-cell need to Be stained with a TCR reactive single chain fragment fret probe for each experiment, transfer about 1 million cells into a tube and spin them down at 400 G.Decant the media and drain remaining media droplets. Sticking to the inner walls with a paper towel. This ensures a minimal incubation volume of about 30 microliters I mentioned earlier.

The fret prope is stored at the concentration of one milligram per milliliter and 50%glycerol at minus 20 degrees Celsius. It is site specifically labeled with either Alexa, flu of 5, 5 5, also three At a dito protein ratio of one at 0.3 microliters of the probe to the cells. Flick the pellet carefully several Times and incubate the cells for 20 minutes on ice.

Wash the cells in ice cold imaging buffer. Repeat this washing procedure once. Incubation, centrifugation storage at low temperature ensure that T cells do not internalize the fluorescent probe while they're exposed to it.

This would result in elevated background fluorescence. Moreover, T cells do not lose the probe in the cold during washing and storage before placing the bilar containing chamber on the microscope stage exchange. The PBS successively with imaging buffer avoid exposure of the bilayer to air at all costs as this would destroy it.

To reduce cellular background, we employ turf illumination. For this, we position the excitation beam into a critical angle at which total internal reflection occurs. Switch off excitation, turn on the wide light and add the T-cell to the ltec chamber and let them settle onto the lipid blay.

Switch on excitation and focus a non turf illumination. The cell membrane is observed as a ring along the optical axis in every focal plane in turf only the basal membrane in contact with the bilayer is observable. Focal planes above are no longer excited and therefore not visible.

The timing protocol starts with the red and green excitation piles for imaging PMHC and TCR followed by a red bleach pulse to ablate the P-M-H-C-F acceptor. A green pulse to image the TCR after F fat acceptor bleaching, and a red pulse to verify F acceptor ablation experiments are performed on a sub-second time scale with the shortest possible delay between the first and the second green pulses. Optionally, A DIC image can be obtained in the beginning two color image splitting allows for the simultaneous detection of the fed donor and fed accepter channel.

The fed accepter excitation shows the accumulation of P HCS in the T-cell synapse. This preble image verifies the presence of pmcs and indicates already TCR mediated PMHC binding.Exciting. The fed donor gives rise to images in both channels.

The signal of the fed donor channel is to some extent reduce in intensity due to energy transfer into the F accepted channel. The signal of the fret accepted channel is composed of fret bleed through of the fed donor and cross excitation of the fret acceptor. Anyhow, this image will not be used for analysis.

In the current protocol. A fast bleach pile follows and eliminates all F accepted fluoro force. The intensity of the F Fed donor image after fret acceptable bleaching is now recovered to higher levels without intensity loss into the fre channel.

The following fret acceptor expectation verifies complete F fat acceptor bleaching. Let's zoom into the synapse to quantify fed donor recovery. Determine the average background intensity a constant regardless of fre acceptor.

Photobleaching then determine the average intensity of the synapse and that of a representative TCR cluster. The fret yield is then calculated by normalizing the difference before and after f acceptor ablation. In our example, we measure 16%fret for the entire synapse and 26%fret for the representative.

TCR cluster fret yields will later be converted into TCR occupancies, which give rise to two dimensional synaptic Dissociation constant single molecule data. Serve Us to deduce the kinetics of synaptic TCR peptide MHC binding for single molecule F threat experiments. We use the same reagents and laboring strategy as before.

However, to ensure that we indeed observe the interaction between a single TCR and a single peptide MHC, we choose to meet the following criterion. A candidate event for a single molecule fre has to colloquially with the single fre acceptor or fre donor molecule. Here we employ a single PMHC as fre acceptor molecule to verify the single molecule nature of the observed fre event, but the opposite rational.

Using fed acceptors in higher buttons and Fred donors and low buttons is certainly an option. We typically expose T cells contacting a stimulatory lipid blay with 514 and 647 nanometer excitation, light sequential and repetitive donor and acceptor excitation with a minimal time lag of one millisecond is followed by delay time. That can be varied according to the requirements of the experiment.

To get a strong enough fluorescent signal for single die detection within milliseconds of excitation, we need to apply high excitation power densities ranging between one and five kilowatts per square centimeter. Due to photobleaching, the number of visible PMHC fret acceptor molecules drops quickly to a level where single fret acceptor fluoro force become distinguishable in a bilayer containing 30 or more labeled PMCS per square micron. An example is shown here for an overall time lag of 42 milliseconds.

A single fret event marked with the red circle becomes visible in the second and third time point. It co localizes with a single S five molecule marked with a green circle. In this example, the observable fret event has a trace length of two, collect more single molecule fret traces as shown in the table in some 128 traces.

Next, calculate the complementary cumulative sum of the traces as shown by the green numbers and normalize them as shown plot normalized values and fit them with a mono exponential function. The expectation value is a little more than 1.5 timeframes. This value depends on TCR peptide, MHC binding and photobleaching to be able to discriminate between these two contributions, the expectation value has thus to be calculated for at least three different time.

Lags now plot all expectation values against their respective time lags and fit the data points to the shown function containing the lifetime here. 2.12 seconds and the expectation value for PHOTOBLEACHING f Fred. Donor recovery after F Fred acceptable bleaching as shown here and in more detail before can be used to determine in just a few steps the two dimensional dissociation constants as they're effective within the immunological synapse.

Here we focus our analysis to TCR engagement and TCR clusters where T-C-R-P-M-H-C binding is most prominent. As becomes evident from the shown measurements F FRED yields vary substantially. For individual TCR clusters.

Multiplying F fred yields with the proportionality factor C gives rise to TCR occupancy, A term describing the ratio between the number of PMHC bound TCRs and their total number C has to be determined once for a specific set of fre pairs and the FRE system employed. Towards the end of this presentation, we'll explain in more detail how to determine C for example, which involves SI three and SI five placed set specifically on the TCR R reactive single chain antibody fragment and the C terminus of the MHC bound peptide. It amounts to about two synaptic, two dimensional dissociation constants can be calculated from the TCR occupancy termed A and the initial density of pmcs present on the lipid blay.

The displayed relationship is derived from the law of mass action and is explained in more detail in the article accompanying this movie. In our example, the initial density of pmcs amounts to 30 molecules per square micron. We arrive at a distribution of 2D kds, which are effective in the T-cell synapse with a median value of about 34 molecules per square micron.

The on rate distribution can be calculated from the synaptic 2D KD distribution and the corresponding off rate here, 0.41 per second as determined via single molecule fret trace analysis at 24 degrees Celsius. The median value is about 0.01 square micron per molecule in second. In summary, F threat measurements between TCR and peptide MHC allow for a comprehensive analysis of all kinetic parameters which describe the reaction dynamics in C two.

This methodology can in principle be generalized to the analysis of other receptor ligand interactions as they might be integral to cell cell recognition. Last but not least, we discuss how to experimentally derive the constant C, which is required to convert fat yields into TCR occupancies. This procedure needs to be performed only once.

The determined constant C can then be employed for all subsequent experiments in which the same fret system with the same fluor fours is applied. In this procedure, we relayed in essence fret yields measured in individual TCR clusters through donor recovery after acceptor photo bleaching to the corresponding intensity values detected in the sensitized fret channel. As discussed before, the fret yield is determined from the background corrected fret donor channel images before and after fret acceptor ablation.

The sensitized fret channel needs to be aligned with the F donor channel and X and Y and corrected for background. Fred donor bleed through and Fred accept cross excitation as shown here. A detailed description of how to correct for Fred donor bleed through and Fred accept cross excitation is provided in the article.

Accompanying this video, now calculate for each TCR cluster the ratio of the bug signal of the corrected fret image and the corresponding corrected fret donor image. Determine the corrected average signal for single fret donors and single molecule fret events and calculate the ratio. The TCR occupancy is then derived by multiplying both ratios.

Now, plot fret yields determined for individual TCR clusters against their TCR occupancies. C is then determined through linear regression as shown here. In Our example, it amounts to a round two.

After watching this video, you Should be able to utilize a protein functionalized lipid bilayer in combination with single molecule fluorescence microscopy to measure the synaptic binding kinetics between PMHC and TCR. All necessary steps including protein production, T-cell labeling, and the final fret measurements were discussed. Furthermore, image analysis was shown in detail yielding T-cell activation curves and fret values.

Finally, the methodology underlying the determination of the kinetic on off rates has been shown. An analogous approach can in principle be used to determine other synaptic receptor ligand interactions, though appropriate modifications might prove to be necessary.