Ab233482

To assess in vivo immunomodulatory effects, tumor
sections from primary tumors gathered across various treatment groups
were subjected to immunofluorescence staining against PD-L1, CD3 or
GrB. In brief, tumor sections were first deparaffinized and then subjected
to heat-mediated antigen retrieval, following a previously published
protocol.^{55 (link)} Subsequently, tumor sections
were blocked with 3% BSA for 30 min and stained overnight at 4 °C
using the following antibodies: anti-PD-L1 antibody (ab233482, 1:100,
Abcam), anti-CD3 antibody (GTX 16669, 1:50, GeneTex, Irvine, CA, USA)
or Anti-GrB antibody (14-8822-82, 1:200, eBioscience, San Diego, CA,
USA). Afterward, the tumor sections were stained with Alexa Fluor
488-conjugated goat antirabbit (ab150077, 1:1000, abcam) for PD-L1
and CD3 or Alexa Fluor 488-conjugated goat antirat antibodies (ab150157,
1:1000, abcam) for GrB, respectively for 1 h at 25 °C. The resulting
images were then captured using a fluorescence microscope (Axio Observer
7).

Human ovarian carcinoma cell lines A2780 and SK-OV-3 were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively. A2780 and SK-OV-3 cells were respectively maintained in RPMI 1640 medium containing 2 mM L-Alanyl-Glutamine (GlutaMAX^TM-I, Gibco, Life Technologies, Saint-Aubin, France) and McCoy’s 5a medium modified (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (FBS). ID8 mouse OC cell line was obtained from Dr. Katherine Roby (University of Kansas Medical Center) [41 (link)]. ID8 cells were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (#D6429, Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 4% FBS, 1% penicillin/streptomycin and insulin–transferrin–sodium selenite media supplement (ITS mix, #I1884, Sigma-Aldrich). Constitutive expression of TSP-1 and PD-L1 by ID8 cell line under unstimulated conditions was ensured by Western blot, using anti-TSP-1 (#ab1823; 1:1000 dilution) and anti-PD-L1 (#ab233482; 3 µg.mL⁻¹) antibodies from Abcam. Cells were cultured in growth medium and split every 3 to 4 days by harvesting in trypsin. Authenticated cell lines by provider were stored at early passages (< 3) in liquid nitrogen, controlled to be mycoplasma-free, and were used in the experiments for no longer than 6 months.

Matrigel domes were broken by mixing them 20 times in 1 mL cold phosphate-buffered saline (PBS) using a P1000 pipette. Content was then transferred into a 15 mL conical tube on ice. Wells were washed with cold PBS (about 1 mL) to capture any remaining organoid pieces. Tubes were centrifugated at 290 g for 5 min at 3–8°C. Once the Matrigel has been eliminated, the supernatant was discarded and RIPA lysis buffer containing protease inhibitor cocktail (#4693116001, Roche) was added. Whole-cell lysates were quantified using a BCA protein assay kit (#23227, ThermoScientific). Twenty micrograms of total protein were used. The blots were probed with primary antibodies against PD-L1 (#ab233482, Abcam) or CTLA4 (#ab237712, Abcam), and beta-actin (#4970, Cell Signaling). ECL-Plus kit was used as a chemiluminescence substrate (#32132, ThermoScientific), and detection was performed using the ChemiDoc MP system (Bio-Rad).

TBI damages the BBB, enabling recruitment of circulating immune cells, such as M/Mɸ, neutrophils, and activated T cells to the injured site. We also took advantage of the injury-induced transient breakdown of the BBB to study whether administration of PD-L1 blocking antibody (Ab) via subcutaneous (SC) injection could reach the injured site of the brain to exert its blocking function. Previous studies have shown that a single dose of anti-PD-L1 Ab (50 μg/kg or 200 μg/kg) via intravenous or intraperitoneal injection can significantly affect stroke outcome through regulating CNS inflammation [37 (link), 38 (link)]. In our study, mice at 24 h post-TBI were subcutaneously injected with either PD-L1 blocking Ab (ab233482, Abcam, Cambridge, MA), PD-L1-Alex 647-conjugated Ab (ab224030, Abcam, Cambridge, MA), or an irrelevant IgG Ab as a control at 200 μg/kg in 100 μL PBS. PD-L1-Alex 647-conjugated Ab was used for histological examination of Ab penetration into the brain, while PD-L1 blocking Ab was used to study the PD-L1 function in the brain. The treatments were blinded to researchers who performed histological evaluations, behavior tests, and flow cytometry.