Pdl 1 13 684

Sequential dual immunofluorescence (IF) was performed on paraffin-embedded tissues mounted onto slides. Tissue sections were labeled for the following antigens: dual CD4/CD8 (NCL-L-CD4-368 and NCL-L-CD8-4B11, Leica Microsystems Inc.), STAT1 (9175, Cell Signaling Technology), CD163 (163 M-18, Cell Marque), PDL-1 (13,684, Cell Signaling Technology), and PD-1 (315 M-96, Cell Marque). This assay was carried out on the Leica Bond Rx fully automated slide staining system (Leica Biosystems) using the Bond Research Detection kit (DS9455). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Heat-induced antigen retrieval was performed at 100ºC in Bond-Epitope Retrieval solution 1 pH-6.0 (AR9961) for 20 or 10 min. After pretreatment, tissues were blocked, and primary antibodies were diluted. Ready-to-use secondary antibodies, Leica’s Novolink Post Primary and/or Novolink Polymer (RE7260-CE) were used, followed by either TSA Cy5 (SAT705A001EA, Akoya Biosciences) or TSA Cy3 (SAT704A001EA, Akoya Biosciences) to visualize the target of interest. Nuclei were stained with Hoechst 33,258 (Invitrogen). The stained slides were mounted with ProLong Gold antifade reagent (P36930, Life Technologies). Positive controls were included for each assay.

The radio immunoprecipitation assay lysis buffer (RIPA) (P0013B, Beyotime, China) containing a protease-inhibitor cocktail (HY-K0010, MCE, USA) were used to extract proteins from cells. The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then 20μg proteins were resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA). The membrane was incubated with primary antibodies against PD-L1(13684, Cell Signaling Technology, USA), FLAG (80010-1-RR, Proteintech, USA), STT3A (12034-1-AP, Proteintech, USA), c-Jun (AF6089, Affinity, USA), HA (51064-2-AP, Proteintech, USA) and GAPDH (60004-1-Ig, Proteintech, USA) at a dilution of 1:1000, followed by incubation with species-specific (rabbit or mouse) HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by Omni ECL reagent (SQ101, EpiZyme, China).

A subset of archived FFPE tumors in the Moffitt cohort were obtained, and PD-L1 expression in each whole tumor slide was determined using PD-L1 #13684 (Cell Signaling Technology). The PD-L1 CPS was determined by counting the number of PD-L1–positive cells (tumor, lymphocytes, and macrophages) divided by the total number of viable cells multiplied by 100 as described previously (22 (link)). In addition, existing PD-L1 CPS data obtained as a standard of care were collected by a retrospective chart review of the Moffitt cohort. For the standard-of-care PD-L1 CPS, the PD-L1 staining was obtained using 22C3 pharmDx assay (Agilent Technologies) as described previously (22 (link)).