Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals

Abstract

Source: Chen, E. W., et al. Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries. J. Vis. Exp. (2019).

In this video, we describe a method to identify the small-molecule kinase inhibitors that modulate the apoptosis of self-reactive CD4⁺CD8⁺ double-positive immature thymocytes. Apoptosis is induced in the double-positive thymocytes by activating them with anti-CD3- and anti-CD28-coated magnetic beads; this is followed by a small-molecule inhibitor treatment and flow cytometry analysis to detect if the inhibitors modulate the apoptotic marker expression.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Kinase Inhibitor Library Screening (Centrifuge-independent Assay)

1. Treatment of thymocytes with kinase inhibitors
   1. Prepare a thymocyte suspension.
   2. Dilute the thymocytes in complete RPMI to obtain a thymocyte suspension of 25 x 10⁶ cells/mL.
   3. Add 40 μL of thymocytes to each well of a small-volume plate, using a multichannel pipette. Place the plate on ice.
   4. Dilute the inhibitors from the stock plate, DMSO, and dexamethasone in complete RPMI at a ratio of four parts of complete RPMI to one part of inhibitor/DMSO/dexamethasone (dilution factor of 5).
      NOTE: As the volumes used in this small-volume plate are 5x smaller than in the conventional method, the inhibitors and the control reagents are diluted fivefold before adding them to the thymocytes in the plate.
   5. Add 0.5 μL of kinase inhibitors to the 96-well plate from the corresponding wells of the inhibitor plate prepared in step 1.1.4.
   6. Prepare eight wells of untreated controls. Prepare four wells of vehicle-treated controls by adding 0.5 μL of the DMSO prepared in step 1.1.4. Prepare four wells of 5 μM dexamethasone-treated controls, using the diluted dexamethasone prepared in step 1.1.4 (Figure 1).

2. Stimulation of thymocytes using anti-CD3/CD28 beads

1. Make sure that the beads are uniformly resuspended. Take 1 mL of beads and wash them with 2 mL of PBS. Separate the beads using a magnetic stand and aspirate the solution. Resuspend the beads in 1 mL of complete RPMI.
   NOTE: The ratio of beads to cells is 1 to 2.5. Adjust the amount of beads, depending on the number of wells to stimulate and the number of thymocytes used.
2. Add 10 μL of bead suspension to each inhibitor-treated sample, the four DMSO-treated samples, and four of the eight untreated samples. Add 10 μL of complete RPMI to the remaining four untreated wells. Figure 1 shows the general plate layout.
   NOTE: The final volume of the wells is 50 μL, which is within the maximum capacity of the wells. It is important to exercise caution and to hold the plates upright, to avoid cross-well spillage.
3. To mix, agitate the plate using a microplate orbital shaker. Alternatively, mix the contents of the wells using a multichannel pipette.
4. Incubate the thymocytes in a 37 °C, 5% CO₂ incubator for 17 - 20 h (or overnight) with an anti-evaporation lid.

3. Setup of the plate washer

NOTE: The instructions for setting up the plate washer are provided by the manufacturer. The steps are mentioned in brief below. Roughly 150 mL of solution is needed for each priming step.

1. Prime the wash system with 70% ethanol containing 1% Tween 20.
2. Prime the wash system with deionized water containing 1% Tween 20.
3. Prime the wash system with FACS wash buffer.

4. Staining of surface antigens

1. Prepare an antibody staining mixture containing anti-TCRβ, anti-CD4, anti-CD8, and anti-CD69 antibodies. Dilute the antibodies in FACS wash buffer at a ratio of 1:100 (v/v).
2. Wash the plate 9x, using 55 μL of FACS wash buffer per wash, using the automated laminar flow washing system.
   NOTE: At the end of the washes, there will be 25 μL of residual volume in each well.
3. Resuspend the cells in 25 μL of the staining antibody mixture prepared in step 1.4.1.
4. If the samples are transferred from a 96-well plate, resuspend the cells in 50 μL of the antibody mixture, and transfer the samples to the small-volume plate. This step corresponds to method number 2, as depicted in Figure 2A.
5. To mix, agitate the plate with a microplate orbital shaker or mix the samples using a multichannel pipette, and incubate on ice for 30 min.

5. Fixation of cells

1. Wash the plate 9x, using 55 μL of FACS wash buffer per wash, using the automated laminar flow washing system.
2. Add fixation/permeabilization buffer (comes with the active caspase-3 apoptosis kit; same with the 10x perm/wash buffer mentioned in step 1.6.1 and the anti-caspase-3 antibody in step 1.6.2) at 50 μL per well.
3. Incubate on ice for 30 min.

6. Intracellular staining for active caspase 3

1. Prepare 1x perm/wash buffer by diluting 25 mL of 10x perm/wash buffer in 225 mL of ultrapure water.
2. Prepare intracellular active caspase stain by adding 1 mL of anti-caspase-3 antibody to 2 mL of 1x perm/wash buffer. The ratio of antibody to perm/wash buffer is 1:2.
3. Prime the wash system with 1x perm/wash buffer.
4. Wash the plate 9x with 1x perm/wash buffer, at 55 μL for each wash.
5. Add 25 μL of the intracellular caspase stain prepared in step 1.6.2 to all wells.
6. To mix, agitate the plate with a microplate orbital shaker or mix the samples using a multichannel pipette, and incubate on ice for 1 h.
7. Wash the plate 9x with 1x perm/wash buffer, at 55 μL for each wash.
8. Add 25 μL of FACS wash buffer to all wells.
9. Transfer the samples to microtiter tubes after adequate mixing via pipetting.
10. Add another 50 μL of FACS wash buffer to the empty wells and repeat step 1.6.9.
11. Repeat steps 1.6.9 and 1.6.10 2x until 200 μL of the samples are collected in the microtiter tubes.
    NOTE: The purpose of the procedures described in steps 1.6.10 and 1.6.11 is to ensure maximum recovery of the cells from the small-volume plate. If cell numbers are not a concern, after step 1.6.10, simply top up the microtiter tubes to 200 μL with FACS wash buffer.
12. Run a flow cytometric analysis of the samples and analyze the results with a FACS analysis program. Caspase-3 activation and CD69 expression are analyzed in the gate containing CD4+CD8+DP thymocytes.

Results

Figure 1: Plate layout of the thymocyte activation assay. (Top) Columns 1 and 12 are reserved for controls, while columns 2 to 11 are inhibitor-treated samples (beige). The negative control (nonstimulated [NS]; grey) occupies wells A1 to D1, and the positive control for cell death (dexamet...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI	HyClone	SH30027FS
FBS	HyClone	SH3007103
L-Glutamine	HyClone	SH3003401
Sodium pyruvate	HyClone	SH3023901
10X PBS	Vivantis	PB0344 – 1L
Kinase Screening Library (96-Well)	Cayman Chemical	10505	Exact contents of the library may vary
DMSO	Sigma Aldrich	D2650
Dexamethasone	Sigma Aldrich	D4902
anti-CD3/CD28 beads	Thermo Fisher Scientific	11452D
FITC Active Caspase-3 Apoptosis Kit	BD Pharmingen	550480	Contains Fixation/Permeabilization buffer, 10X Perm/Wash buffer and anti-caspase 3 antibody
DA-Cell Washer	CURIOX	HT1000
96-well DA-Cell Plate	CURIOX	96-DC-CL-05
Antibodies
CD3e	BioLegend	100236
TCRb	BD Biosciences	553174
CD4	BD Biosciences	740007
CD8	BD Biosciences	563786
CD69	eBioscience	25-0699-42
Inhibitors
TG003	Cayman Chemical		From the Kinase Screening Library
TG003	Cayman Chemical	-	From the Kinase Screening Library
PKC 412	Cayman Chemical	-	From the Kinase Screening Library
Doramapimod	Cayman Chemical	-	From the Kinase Screening Library
Paclitaxel	Cayman Chemical	-	From the Kinase Screening Library
Erlotinib	Cayman Chemical	-	From the Kinase Screening Library
Necrostatin-5	Cayman Chemical	-	From the Kinase Screening Library
NVP-BEZ235	Cayman Chemical	-	From the Kinase Screening Library
Phthalazinone pyrazole	Cayman Chemical	-	From the Kinase Screening Library
AG-879	Cayman Chemical	-	From the Kinase Screening Library
1-NA-PP1	Cayman Chemical	-	From the Kinase Screening Library
Torin 1	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide II	Cayman Chemical	-	From the Kinase Screening Library
BIBF 1120	Cayman Chemical	-	From the Kinase Screening Library
SMI-4a	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide XI (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
CAY10657	Cayman Chemical	-	From the Kinase Screening Library
AS-703026	Cayman Chemical	-	From the Kinase Screening Library
Chelerythrine chloride	Cayman Chemical	-	From the Kinase Screening Library
Tunicamycin	Cayman Chemical	-	From the Kinase Screening Library
GSK 1059615	Cayman Chemical	-	From the Kinase Screening Library
Ruxolitinib	Cayman Chemical	-	From the Kinase Screening Library
Necrostatin-1	Cayman Chemical	-	From the Kinase Screening Library
SB 505124	Cayman Chemical	-	From the Kinase Screening Library
INK128	Cayman Chemical	-	From the Kinase Screening Library
Canertinib (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
SB 431542	Cayman Chemical	-	From the Kinase Screening Library
PD 173074	Cayman Chemical	-	From the Kinase Screening Library
Valproic Acid (sodium salt)	Cayman Chemical	-	From the Kinase Screening Library
PD 0325901	Cayman Chemical	-	From the Kinase Screening Library
SB 203580	Cayman Chemical	-	From the Kinase Screening Library
VX-702	Cayman Chemical	-	From the Kinase Screening Library
Emodin	Cayman Chemical	-	From the Kinase Screening Library
CHIR99021	Cayman Chemical	-	From the Kinase Screening Library
BIO	Cayman Chemical	-	From the Kinase Screening Library
Imatinib (mesylate)	Cayman Chemical	-	From the Kinase Screening Library
Sunitinib Malate	Cayman Chemical	-	From the Kinase Screening Library
Gefitinib	Cayman Chemical	-	From the Kinase Screening Library
PP2	Cayman Chemical	-	From the Kinase Screening Library
3-Methyladenine	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide I	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide IV	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide V	Cayman Chemical	-	From the Kinase Screening Library
NSC 663284	Cayman Chemical	-	From the Kinase Screening Library
D 4476	Cayman Chemical	-	From the Kinase Screening Library
NU 7026	Cayman Chemical	-	From the Kinase Screening Library
H-9 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
Indirubin-3'-monoxime	Cayman Chemical	-	From the Kinase Screening Library
KN-62	Cayman Chemical	-	From the Kinase Screening Library
KN-93	Cayman Chemical	-	From the Kinase Screening Library
CGP 57380	Cayman Chemical	-	From the Kinase Screening Library
Iso-Olomoucine	Cayman Chemical	-	From the Kinase Screening Library
(S)-Glycyl-H-1152 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
Bisindolylmaleimide VIII (acetate)	Cayman Chemical	-	From the Kinase Screening Library
ST638	Cayman Chemical	-	From the Kinase Screening Library
SU 6656	Cayman Chemical	-	From the Kinase Screening Library
LY364947	Cayman Chemical	-	From the Kinase Screening Library
SB 203580 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
CAY10621	Cayman Chemical	-	From the Kinase Screening Library
YM-201636	Cayman Chemical	-	From the Kinase Screening Library
ZM 447439	Cayman Chemical	-	From the Kinase Screening Library
AS-041164	Cayman Chemical	-	From the Kinase Screening Library
NVP-AEW541 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
PP242	Cayman Chemical	-	From the Kinase Screening Library
ABT-869	Cayman Chemical	-	From the Kinase Screening Library
CAY10622	Cayman Chemical	-	From the Kinase Screening Library
17β-hydroxy Wortmannin	Cayman Chemical	-	From the Kinase Screening Library
CAY10626	Cayman Chemical	-	From the Kinase Screening Library
SU 6668	Cayman Chemical	-	From the Kinase Screening Library
CAY10572	Cayman Chemical	-	From the Kinase Screening Library
N,N-Dimethylsphingosine	Cayman Chemical	-	From the Kinase Screening Library
LY294002	Cayman Chemical	-	From the Kinase Screening Library
U-0126	Cayman Chemical	-	From the Kinase Screening Library
Staurosporine	Cayman Chemical	-	From the Kinase Screening Library
KN-92 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
AS-605240 (potassium salt)	Cayman Chemical	-	From the Kinase Screening Library
O-1918	Cayman Chemical	-	From the Kinase Screening Library
Y-27632 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
Leelamine	Cayman Chemical	-	From the Kinase Screening Library
PD 98059	Cayman Chemical	-	From the Kinase Screening Library
PD 169316	Cayman Chemical	-	From the Kinase Screening Library
TGX-221	Cayman Chemical	-	From the Kinase Screening Library
(S)-H-1152 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
AS-605240	Cayman Chemical	-	From the Kinase Screening Library
D-erythro-Sphingosine C-18	Cayman Chemical	-	From the Kinase Screening Library
OSU03012	Cayman Chemical	-	From the Kinase Screening Library
JNJ-10198409	Cayman Chemical	-	From the Kinase Screening Library
Leelamine (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
Arachidonic Acid Leelamide	Cayman Chemical	-	From the Kinase Screening Library
Lauric Acid Leelamide	Cayman Chemical	-	From the Kinase Screening Library
AS-252424	Cayman Chemical	-	From the Kinase Screening Library
CAY10505	Cayman Chemical	-	From the Kinase Screening Library
PI-103	Cayman Chemical	-	From the Kinase Screening Library
PIK-75 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
Sphingosine Kinase Inhibitor 2	Cayman Chemical	-	From the Kinase Screening Library
Piceatannol	Cayman Chemical	-	From the Kinase Screening Library
SC-1	Cayman Chemical	-	From the Kinase Screening Library
(R)-Roscovitine	Cayman Chemical	-	From the Kinase Screening Library
BAY-43-9006	Cayman Chemical	-	From the Kinase Screening Library
CAY10561	Cayman Chemical	-	From the Kinase Screening Library
AS-604850	Cayman Chemical	-	From the Kinase Screening Library
PI3-Kinase α Inhibitor 2	Cayman Chemical	-	From the Kinase Screening Library
ML-9	Cayman Chemical	-	From the Kinase Screening Library
Triciribine	Cayman Chemical	-	From the Kinase Screening Library
Erbstatin Analog	Cayman Chemical	-	From the Kinase Screening Library
Kenpaullone	Cayman Chemical	-	From the Kinase Screening Library
Olomoucine	Cayman Chemical	-	From the Kinase Screening Library
AG-494	Cayman Chemical	-	From the Kinase Screening Library
AG-825	Cayman Chemical	-	From the Kinase Screening Library
AG-1478	Cayman Chemical	-	From the Kinase Screening Library
SB 216763	Cayman Chemical	-	From the Kinase Screening Library
SB 415286	Cayman Chemical	-	From the Kinase Screening Library
AG-17	Cayman Chemical	-	From the Kinase Screening Library
H-8 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
LFM-A13	Cayman Chemical	-	From the Kinase Screening Library
SC-514	Cayman Chemical	-	From the Kinase Screening Library
Apigenin	Cayman Chemical	-	From the Kinase Screening Library
AG-18	Cayman Chemical	-	From the Kinase Screening Library
CAY10554	Cayman Chemical	-	From the Kinase Screening Library
DRB	Cayman Chemical	-	From the Kinase Screening Library
RG-13022	Cayman Chemical	-	From the Kinase Screening Library
RG-14620	Cayman Chemical	-	From the Kinase Screening Library
AG-490	Cayman Chemical	-	From the Kinase Screening Library
AG-82	Cayman Chemical	-	From the Kinase Screening Library
AG-99	Cayman Chemical	-	From the Kinase Screening Library
AG-213	Cayman Chemical	-	From the Kinase Screening Library
AG-183	Cayman Chemical	-	From the Kinase Screening Library
Lavendustin C	Cayman Chemical	-	From the Kinase Screening Library
ZM 336372	Cayman Chemical	-	From the Kinase Screening Library
5-Iodotubercidin	Cayman Chemical	-	From the Kinase Screening Library
SB 202190	Cayman Chemical	-	From the Kinase Screening Library
CAY10571	Cayman Chemical	-	From the Kinase Screening Library
Nilotinib	Cayman Chemical	-	From the Kinase Screening Library
SP 600125	Cayman Chemical	-	From the Kinase Screening Library
L-threo-Sphingosine C-18	Cayman Chemical	-	From the Kinase Screening Library
H-89	Cayman Chemical	-	From the Kinase Screening Library
HA-1077 (hydrochloride)	Cayman Chemical	-	From the Kinase Screening Library
AG-370	Cayman Chemical	-	From the Kinase Screening Library
Wortmannin	Cayman Chemical	-	From the Kinase Screening Library
AG-1296	Cayman Chemical	-	From the Kinase Screening Library
KT 5823	Cayman Chemical	-	From the Kinase Screening Library
Janex 1	Cayman Chemical	-	From the Kinase Screening Library
CAY10574	Cayman Chemical	-	From the Kinase Screening Library
CAY10575	Cayman Chemical	-	From the Kinase Screening Library
CAY10576	Cayman Chemical	-	From the Kinase Screening Library
NH125	Cayman Chemical	-	From the Kinase Screening Library
TWS119	Cayman Chemical	-	From the Kinase Screening Library
NSC 210902	Cayman Chemical	-	From the Kinase Screening Library
CAY10577	Cayman Chemical	-	From the Kinase Screening Library
CAY10578	Cayman Chemical	-	From the Kinase Screening Library
PD 184161	Cayman Chemical	-	From the Kinase Screening Library
CCT018159	Cayman Chemical	-	From the Kinase Screening Library
Myricetin	Cayman Chemical	-	From the Kinase Screening Library