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

Published: August 31, 2023

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.

Representative Results

Figure 1
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 (dexamethasone-treated [DEX]; purple) occupies wells E1 to H1. Columns 2 to 12 contain thymocytes stimulated with anti-CD3/CD28 beads. The positive control for thymocyte activation (stimulated samples [α-CD3/CD28]; green) occupies wells A12 to D12, and the vehicle control (stimulated and DMSO-treated [α-CD3/CD28 + DMSO]; red) occupies wells E12 to H12. (Bottom) Flow cytometry plots of active caspase-3 (ActCasp3), CD69, and TCRβ staining of thymocytes gated within the double-positive (DP) gate. Representative plots of the different controls are shown. NS = nonstimulated; DEX = dexamethasone-treated samples; α-CD3/CD28 + DMSO = samples stimulated with CD3/CD28-coated beads and treated with DMSO; α-CD3/CD28 = samples stimulated with CD3/CD28-coated beads.

Figure 2
Figure 2: Thymocyte viability after treatment with inhibitors. (A) Experimental outline of the major steps in the screening assay. There are three proposed methods for the stimulation and staining of the thymocytes used in the activation assay, namely (1) the culturing of thymocytes in standard 96-well plates, followed by staining using a conventional centrifugation-based protocol, (2) the culturing of thymocytes in standard 96-well plates, followed by staining using a centrifugation-independent washing protocol, and (3) the culturing of thymocytes in small-volume plates, followed by staining in the same plates using a centrifugation-independent washing protocol. (B) Gating strategies used in the viability assays. The live cell gate was derived from the forward scatter (FSC) and side scatter (SSC) plots. Inhibitors that were deemed to be too toxic at the tested concentration were subject to further viability assays at 10-fold lower concentrations. Representative inhibitor-treated samples are shown. Note the common control (DMSO-treated [DMSO]) used for the 1 µM and 0.1 µM samples. (C) Plate layout of diluted inhibitors. A schematic representation of the plates of inhibitors diluted in DMSO to a concentration of 500x the intended final concentration. Each well represents one unique inhibitor; the grey wells are empty. The concentrations shown are the final concentration when added to the cell cultures, namely 10 µM (dark red), 1 µM (fuchsia), and 0.1 µM (blue).

Declarações

The authors have nothing to disclose.

Materials

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

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Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals. J. Vis. Exp. (Pending Publication), e21605, doi: (2023).

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