Imipramine

To assess the cytotoxicity of ESO CTLs, MCF-7 breast cancer cells were transduced with NY-ESO-1 and CLEC2D lentiviral vectors (NY-ESO-1⁺ CLEC2D⁺ MCF-7). NY-ESO-1⁺CLEC2D⁺ MCF-7 cells were labeled by CellTracker CMFDA Dye (Thermo Fisher Scientific, #C7025) and passed through Dead Cell Removal Microbeads (Miltenyi Biotec, #130-090-101), then cocultured with ESO CTLs at an effector/target ratio of 1:1 for 8 hours. For the cytotoxicity of TILs, primary breast cancer cells were isolated from tumor tissue and purified by EpCAM Microbeads (Miltenyi Biotec, #130-061-101). Then celltracker-labeled primary tumor cells were passed through Dead Cell Removal Microbeads and cocultured with autologous pentamer⁺ CTLs (CTLs isolated by FACS using MUC1 pentamer) at an effector/target ratio of 1:1 and autologous CD161⁺ CTLs at an effector/target ratio of 10:1 for 12 hours. In some experiments, CD161⁺ and CD161^– T cells were separated and seeded into 96-well plates via FACS (BD Influx). At the end of the coculture, all cells were stained with PI (eBioscience, # 00-6990, 1:50) and analyzed by flow cytometry immediately. In some experiments, T cells were preincubated with imipramine (Sigma-Aldrich, #I0899, 100 μmol/L) for 1 hour before coculturing with autologous tumor cells.

T cells were lysed in lysis buffer (50 mmol/L sodium acetate [pH 5], 0.5% Nonidet P-40). 10 μg cell lysates from T cells was incubated with 0.2 mg sphingomyelin (Sigma-Aldrich, # 85615, dissolved in a 1:1 mixture of chloroform and methanol) for 2 hours and then loaded 30 μL onto a silica gel 60 thin-layer chromatography plate (EMD Millipore, #99570) along with known concentrations of ceramide (natural ceramide, APExBIO, #A4534). Then the thin-layer chromatographic separation was performed using a solvent system of chloroform and methanol (3:2) for 40 to 80 minutes until the solvent front was 1 to 2 cm from the top. Ceramide was visualized in iodine vapor and scanned by a high-resolution gel imaging system (Bio-Rad, Gel Doc XR). ImageJ software (NIH) was used to evaluate relative ceramide levels. In some experiments, T cells were preincubated with imipramine (Sigma-Aldrich, # I0899, 100 μmol/L) for 1 hour before microbead-mediated CD161 cross-linking (see “Intracellular Ca²⁺ measurements”) and ceramide production measurements.

Male C57BL/6J mice (8- to 10-week-old at the start of experiments) were obtained
from Changzhou Cavens Laboratory Animal Co. Ltd (Changzhou, China). Animals were
hosted on a 12 h light/dark cycle (lights on at 6:00 am and off at 6:00
pm) under controlled temperature (22 ± 2°C) and humidity (50 ±
10%), with standard diet and water ad libitum. Animals were acclimatized for 7
days. The experimental procedures had been approved by the Animal
Experimentation Ethics Committee of China Pharmaceutical University and under
the guidelines of “Principles of Laboratory Animal Care” (NIH publication No.
80-23, revised in 1996). All efforts were made to minimize suffering.
Sucrose adaptation and sucrose consumption assessment were performed to the CMS
procedures at the beginning of the experiment trial. The CMS procedure was
carried out with some adjustments. Briefly, a series of stressors were applied
onto the animals: (1) water deprivation for 24 h, (2) stroboscopic illumination
for 2 h, (3) cage tilt (45°) for 15 h, (4) noise for 2 h, (5) soiled cage
(200 mL water in 100 g sawdust bedding) for 15 h, (6) body restraint for 1 h,
(7) forced swimming at 8°C for 6 min, (8) tail-clipping restraint for 6 min, (9)
food deprivation for 24 h, and (10) day and night reverse. These stressors were
randomly arranged in 1 week and repeated for 6 weeks. At the end of the CMS
procedure, a sucrose preference test was carried out to evaluate the CMS
model.^{23 (link)}The mice were randomly divided into 5 groups (n = 12). The control and CMS model
were given with saline. For the other 3 groups, AC at low dose (50.0 mg/kg/day),
high dose (150 mg/kg/day), and imipramine (30 mg/kg/day) were intragastrically
given 30 min before stress exposure for 6 weeks. Mouse body weight was recorded
twice a week during CMS and AC treatment. The mice were weighed using an
electronic scale and the average of two measurements was recorded.
Sucrose preference test was conducted out at the end of the CMS procedure. In
brief, mice in each group were learned to adapt to 2 bottles of 1% sucrose
solution (w/v) 72 h before the test, and 24 h later, one bottle of 1% sucrose
solution (w/v) was replaced with tap water for 24 h. Then, mice were deprived of
water and food for 24 h. Sucrose preference test was conducted at 17:00
pm, where mice were kept in individual cages with 2 bottles, one
with 100 mL of 1% sucrose solution (w/v) and the other with 100 mL of water.
After 3 h, the volumes of consumed sucrose solution and water were recorded and
the sucrose preference was calculated by the following formula: sucrose
preference = sucrose consumption/ (water consumption + sucrose consumption) ×
100%.^{24 (link)}Forced swimming test was carried out at the end of CMS procedures. Mice in each
group were placed in large glass cylinders (50 cm height and 20 cm diameter)
with 30 cm height water at 22 ± 2°C, so that mice were not able to support
themselves by hind limbs. The test consists of 2 parts: the first 15 min was
used for pre-swimming and then 24 h later, swimming behavior was observed for
5 min, and the latency to float was measured and analyzed.^{25 (link)}