Our high-throughput assay adds to the available methods for the identification of small molecules and their targets that modulate TCR signaling and T-cell activation. Our assay has a self-corrective aspect through filtering out toxic compounds and identifying inhibitors of TCR signaling and stimulation-induced apoptosis. Identification of mediators of TCR signaling can aid in the developments of therapy for immune diseases.
We provided a strong stimulus for thymocytes derived from TCR-polyclonal mice. It is possible to use TCR-transgenic mice and stimulate them with other peptide ligand tetramers for weaker stimulation. This assay involves the use of small volumes and of small-volume plates for culturing of the cells.
This necessitates careful handling of the plates and its contents. There are many compounds in the library of kinase inhibitors which are considered hazardous. For safety considerations, treat all compounds as equally hazardous, and follow the supplier's recommended safety precautions.
To begin the treatment of thymocytes with kinase inhibitors, use a multi-channel pipette to add 40 microliters per well of thymocytes to a small-volume plate. Put the plate on ice. Then pipette one part of kinase inhibitors, DMSO, and dexamethasone, and four parts of the complete RPMI media into a 96-well plate.
Designate eight wells of the small-volume plate to untreated controls, four wells to dexamethasone-treated positive controls for cell death by adding the diluted dexamethasone at the five-micromolar concentration, and four wells to vehicle-treated controls, by adding 0.5 microliters of the diluted DMSO. Next, from the corresponding wells of the inhibitor plate, add 0.5 microliters of each diluted inhibitor to the 96-well plate. To begin thymocyte stimulation, first make sure anti-CD-3 and CD-28 beads are uniformly re-suspended.
Next, wash one milliliter of the beads with two milliliters of the PBS buffer. Put the tube on a magnetic stand to separate beads from supernatant and aspirate the solution. Then re-suspend the beads in one milliliter of the complete RPMI medium.
Add 10 microliters per well of the bead suspension to each inhibitor-treated sample, the four DMSO-treated samples, and four of the eight untreated samples. Add 10 microliters of the complete RPMI to the remaining four untreated wells. Use a microplate orbital shaker to gently agitate the plate.
Next, incubate the thymocytes in 37 degrees Celsius and 5%carbon dioxide environment for 17 to 20 hours, or overnight. Prime an automated laminar flow plate washer, first with 150 milliliters of ethanol Tween solution, then with deionized water supplemented with 1%Tween 20, and finally, with FACS wash buffer. At the end of the incubation using the plate washer system, wash the plate with FACS wash buffer using a nine-times washing cycle.
Each wash adds and removes 55 microliters of wash buffer by laminar flow, resulting in an exponential dilution of the reagents in the wells. To stain surface antigens, first dilute one unit volume of anti-CD-3, anti-CD-4, anti-CD-8, and anti-CD-69 antibodies in 100-unit volumes of the FACS wash buffer. Then re-suspend the cells in 25 microliters of the staining antibody mixture.
Use a microplate orbital shaker to gently agitate the plate, and then leave the plate on ice for 30 minutes. To fix the cells, first wash the plate with 55 microliters of FACS wash buffer using nine-times washing cycle as described before. Then add 50 microliters of the fixation-impermeablization buffer to each well.
Mix well and incubate the plate on ice for 30 minutes. After the incubation, prepare a one-X perm and wash buffer by diluting 25 milliliters of the provided 10X stock in 225 milliliters of Ultrapure water. Prime the plate washer with one-X buffer and wash the plate with 55 microliters of the one-X buffer using a nine-times washing cycle as described before.
To begin, mix one milliliter of the anti-caspase 3 antibody with two milliliters of the one-X perm and wash buffer. Add 25 microliter per well of the mixture to the fixed cells. Use a microplate orbital shaker to gently agitate the plate.
Leave the plate on ice for an hour. At the end of incubation, repeat the wash step nine times with the one-X perm and wash buffer. Then add 25 microliters of the FACS wash buffer to all wells of the plate.
Pipette up and down a few times to mix the solution. Finally, transfer the mixed samples into microtiter tubes. Top up the tubes with the FACS wash buffer to a total volume of 200 microliters, and proceed to the flow cytometry analysis.
Following anti-CD-3, CD-28 stimulation, an increase in caspase-3 activation and the TCR downregulation were observed in both the untreated and the DMSO mock-treated samples. Also, an increase in CD-69 expression was observed in both stimulated samples. The dexamethasone-treated samples showed an increase in caspase-3 activation independent of CD-69 upregulation, as expected for the independent apoptosis-inducing effect and the TCR stimulation.
The selective impairment of T-cell activation phenomena was shown by different inhibitors that either suppressed both caspase-3 activation and CD-69 up-regulation, or inhibited CD-69 up-regulation, but did not impair caspase-3 activation. There were also inhibitors that did not target a relevant kinase of the TCR-signaling pathway, and therefore did not suppress both CD-69 upregulation and caspase-3 activation. The screen result of staurosporine, the apoptosis-positive control, showed high levels of caspase-3 activation, as expected.
However, the result also showed low levels of CD-69 expression that can be attributed to either its mediated inhibition of protein kinase C or its induced apoptosis before CD-69 expression. Comparison of different assay protocols showed no differences in active caspase-3, CD-69, and TCR staining of the negative control, the positive control for cell death, the vehicle control, and an inhibitor-treated sample. The pre-incubation stages are meant to facilitate the use of the small-volume plate for the culturing of the cells.
The standard centrifuge-dependent protocol is also provided in the manuscript. Potentially-interesting inhibitors or targets can be validated using different cell types and also in different species, such as pagopheral mouse lymphocytes or human primary cells. Subsequent characterization of the identified molecules and the targets can help improve understanding of T-cell biology and expand on possible targets for immunomodulation.
