Isotype control

MDM were infected with Streptococcus pneumoniae strain D39 serotype 2 at an MOI of 0.1 in RS-RPMI (without antibiotics) for 2 h in the presence or absence of 10 μg/ml CD36 blocking antibody or isotype control (Abcam, Cambridge, UK). MDMs were washed and incubated for 30 min in antibiotic-containing RS-RPMI to remove extracellular bacteria. Cells were then incubated in 1X Permwash (BD) for 20 min before vortexing and plating onto blood agar.

The cells at passage 3 with good growth state were detached and then prepared for single cell suspension with phosphate buffer saline (PBS) containing 1% bovine serum albumin (BSA). About 1 × 10⁶ cells/l00 μL were packed into several Eppendorf (EP) tubes, the mesenchymal stem cells that were packed into each EP tube were added with fluorescent-labeled antibody: FITC-CD45, PE-CD29, FITC-CD90, PE-CD34, PE-CD105 and isotype control (Abcam Cambridge, MA, USA), respectively. The cells were dissolved on ice for 1 h, then washed with PBS containing 1% BSA for 3 times, and then suspended again with PBS. Make sure that there were at least 1 × 10⁵ cells for detection per tube. A flow cytometer was used for detection (Becton, Dickinson and Company, Franklin lake, New Jersey, USA).

Immunohistochemistry was carried out using the Histostain‐Plus 3rd Gen IHC Detection Kit (Invitrogen, Carlsbad, CA). The sections of knee joints were de‐paraffinized and rehydrated through conventional methods. Endogenous peroxidase was blocked by treating the sections with 3% hydrogen peroxide in methanol for 30 min. The sections were digested by 100 mg/ml hyaluronidase (Sigma, St. Louis, MO) for 15 min. Nonspecific protein binding was blocked by incubation with a serum blocking solution. The sections were then immunostained using antibodies against ADAMTS‐5, NOTCH1, and COL II (Abcam, Cambridge, MA), AGGRECAN neoepitope antibody representing the neoepitope sequence (NITEGE) generated by aggrecanase‐mediated cleavage at Glu ³⁷³‐Ala³⁷⁴ of AGGRECAN core proteins (Novus biologicals), and IL‐6 (Invitrogen, Carlsbad, CA), respectively, at 4°C overnight. The negative control sections were incubated with isotype control (Abcam) in 0.01m PBS. Thereafter, the sections were treated sequentially with ready‐to‐use biotinylated secondary antibody and ready‐to‐use streptavidin–peroxidase conjugate, followed by standardized development in DAB chromogen. The sections were counterstained with ready‐to‐use hematoxylin (Invitrogen). Images are captured at 40× magnification with a Nikon E800 microscope (Melville, NY).

Freshly collected blood samples were treated with Fc block (Miltenyi Biotech) and then subjected to red blood cell lysis (Thermo Fisher Scientific, eBioscience RBC Lysis Buffer). After spinning and washing with 2% FBS in PBS, cells were subjected to surface staining. For freshly collected effusion samples, the cells were subjected to red blood cell lysis after spinning, passed through a 40-μm cell strainer (FALCON), and stained. To detect nivolumab binding, samples were incubated with anti–human IgG4-PE or isotype control (Abcam) after Fc blocking. Antibody clone numbers for immune analysis are listed in the Supplemental Methods. Fixation/permeabilization buffers (Thermo Fisher Scientific, eBioscience Foxp3/Transcription Factor Staining Buffer Set) were used for intracellular staining.

Cells were prepared as single-cell suspensions. They were conjugated with rabbit anti-rat LIGHT primary antibody (Abcam) or isotype control (Abcam) and then with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen). Afterwards the cells were combined with APC A750-rat CD45 (eBioscience, San Diego, CA, USA) or isotype control (eBioscience). Finally, flow cytometry was performed with a Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter, Miami, FL, USA), and the results were analyzed with FlowJo software.

For double immunofluorescence staining, 5 µm thick lung sections were incubated overnight with a mixture of rabbit anti‐CD248 and anti‐α‐SMA mouse antibody, and hPASMCs were incubated overnight with a mixture of rabbit anti‐CD248 and anti‐PDGFR‐β mouse antibody at 4°C; instead, the antibody was substituted with isotype control (Abcam). The images were received using SP5II microscopy and were analyzed using Image J. To detect hPASMC apoptosis, cells were observed using a TUNEL assay, with a one‐step TUNEL apoptosis kit (MA0224, Meilunbio, Dalian, China), and were counterstained with DAPI.