Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

The overall goal of this procedure is to generate soluble recumbinate T-Cells receptor and peptide HLA proteins of sufficient quality for use in biophysical and structural analysis. This method could help answer questions in the field of T-Cell Immunology. Such as, for example, why do T-Cells not respond robustly to cancer?

The main advantage of this technique is that we get extremely high resolution mechanistic data explaining how T-Cell receptors recognize their targets, leading to T-Cell mediated immune responses. The implications of this technique extend toward therapy or diagnosis of virtually any human disease because T-Cells play a pivotal role in most immune responses. Though this method can provide insight into T-Cell immune responses, it can also be applied to other systems that are controlled by receptor ligand interactions.

Generally, individuals new to this method will struggle because it is very difficult to generate soluble, active proteins of sufficient quality for biophysical and structural experiments. To begin the peptide HLA refolding procedure to six milliliters of guanidine buffer with 10 milometer dithiothreitol, add 30 milligrams of HLA-A2 inclusion bodies, 30 milligrams of beta 2-M inclusion bodies and four milligrams of the desired peptide of interest. Incubate the mixture for 30 minutes at 37 degrees Celsius.

Then, dilute the mixture with one liter of HLA refold buffer, pre-chilled to four degrees Celsius and stir the mixture for three hours at that temperature. Dialyze the mixture twice against 20 liters of 10 millimolar tris, pH 8.1, for 24 hours each. To begin the TCR refolding procedure, to six milliliters of guanidine buffer with 10 millimolar dithiothreitol, add 30 milligrams each of TCR Alpha and beta chain IB's.

Incubate the mixture for 30 minutes at 37 degrees Celsius and dilute the mixture in one liter of pre-chilled TCR refold buffer. Stir and dialyze the diluted TCR refold mixture in the same way as the peptide HLA mixture. Filter both dialyzed refold mixtures through a 0.45 micrometer membrane filter.

Next, load one of the filtered refold preparations onto a 7.9 milliliter 50 micrometer anion exchange resin column, pre-equilibrated with 20 milliliters of 10 millimolar tris, pH 8.1. Elute the protein with a salt gradient buffer system at five milliliters per minute. Collect one milliliter fractions and analyze the fractions with SDS-Page.

Combine the fractions containing the refolded protein. Place the protein mixture in a 10 kilodalton molecular weight cutoff centrifugal concentrator. Centrifuge the mixture for 20 minutes or however long is required, at 4000 times g, to concentrate the mixture to 500 microliters.

Discard the flow through. Mold the concentrated protein preparation into a two milliliter injection loop and inject the protein onto a 24 milliliter size exclusion chromatography column, pre-equilibrated with the appropriate elusion buffer. Elute the protein with one column volume of buffer at 0.5 milliliters per minute.

Collect one milliliter fractions, analyze the fractions with SDS-Page and combine the fractions containing the protein of interest. Prepare 10 serial dilutions of the TCR solution, in a concentration range spanning 10 times above to 10 times below the KD of the specific TCR peptide HLA interaction if known. Otherwise, start from a TCR concentration of 200 micromolar.

Next, activate a carboxymethylated dextran-coated sensor chip by flowing a one to one mixture of 100 millimolar and N-Hydroxysuccinimide and 400 millimolar EDC over the chip at a flow rate of 10 microliters per minute for 10 minutes, at 25 degrees Celsius. Then, load 110 microliters of 200 micrograms per milliliter streptavidin and 10 millimolar acetate pH 4.5 in all four flow cells to functionalize the chip surface. Deactivate any remaining reactive groups with 100 microliters of one molar ethanolamide hydrochloride.

Next, flow a solution of approximately one micromolar peptide HLA in the chip manufacturer-provided buffer over the chip's surface at 10 microliters per minute until the response unit count has an increase by 500 to 600. Saturate the chip's surface with one millimolar biotin and manufacture-provided buffer for 60 seconds. Then, inject the 10 serial dilutions of the TCR solution at 30 microliters per minute at 25 degrees Celsius.

Calculate the equilibrium binding constant and run a kinetics analysis. Perform a series of SPR experiments in this way, varying only the temperature at which the TCR dilutions are injected. Determine the equilibrium binding constant for each temperature and calculate the Gibbs energies and other thermodynamic parameters.

Plug the Gibbs energies against temperature using a non-linear regression fit. Concentrate the peptide HLA solution to 0.2 millimolar and crystallization buffer by centrifugation at 3, 000 times g, in a 10 kilodalton molecular weight cutoff centrifugal concentrator. Prepare a 0.1 millimolar solution of one to one TCR and peptide HLA for co-complex crystallization.

Using a crystallization robot and a screening kit, prepare crystallization trials using the sitting drop technique in a 96-Well plate. With 60 microliters of crystallization buffer in each reservoir, and each drop being composed of 200 nanoliters each of protein solution and buffer. Grow the crystals at 18 degrees Celsius.

Score for crystals under a microscope after 24 hours, 48 hours, 72 hours, and then, weekly. Once single crystals of sufficient quality have grown, harvest the single crystals with a cryogen-compatible loop and store the crystals in liquid nitrogen. Perform synchrotron x-ray diffraction under a stream of nitrogen gas at 100 kelvin, analyze the data and solve the structures with the appropriate software.

Calculate the contacts, surface complementarity and the crossing angle from the solved structures. Using these methods, TCR and peptide HLA molecules were expressed and refolded into a soluble form. The peptide in the peptide HLA molecules was the gp100 antigen.

SPR was used to determine the binding affinity of the PMEL17 TCR for the antigen at various temperatures. SPR and isothermal calorimetric titration were then both used, to determine the thermodynamic parameters of the protein-protein interaction. The PMEL17 TCR A2-YLE-9V complex was crystallized and evaluated with synchrotron x-ray diffraction.

The TCR CDR loops and the peptide HLA residues contacted by the TCR protein, were both identified and the nature of the contacts was studied. Peptide HLA molecules with mutated forms of the peptide were also purified and crystallized in this way. To investigate structural differences that could prevent binding interactions with TCR.

Mutation of the third or fifth residue was found to significantly change the position of proline four, which would disrupt many of the contacts with the TCR CDR-3 Alpha Loop. Whilst attempting this procedure, it's important to select only the purest proteins for further analysis. Following this procedure, other methods like, peptide HLA tetramer staining, can be performed using the soluble proteins in order to isolate antigen-specific T-Cells from patient blood for diagnostics and further analysis.

After its development, this technique paved the way for researchers in the field of T-Cell immunology to explore TCR peptide HLA binding in exquisite detail, enabling the development of new medicines for cancer and other human diseases. After watching this video, you should have a good understanding of how to generate TCR and peptide HLA proteins of sufficient quality to perform biophysical and structural analysis.