Anti icos

A total of 16 human LUAD tissue samples were obtained from Tianjin Medical University Cancer Institute and Hospital and informed consent was provided by all patients. Total protein was prepared in RIPA buffer (Solarbio, Beijing, China). Each sample was separated by a 10% SDS‐PAGE gel and transferred to a PVDF membrane. After blocking for two hours with 5% BSA, membranes were incubated overnight at 4°C with the primary antibodies, anti‐CD63 (Santa Cruz), anti‐tsg101 (Santa Cruz), anti‐ICOS (Abcam), and anti‐β‐actin (ZSGB‐BIO, China). The blots were incubated with secondary antibodies (ZSGB‐BIO, China) (1:2000) for one hour at room temperature, followed by detection with ECL reagents (Pierce). Protein levels were quantified using ImageJ software.

CD4⁺ T cells were lysed in protein lysis buffer containing proteinase inhibitor (Thermo Fisher Scientific). The lysates were centrifuged for 15 min at 14,000 × g at 4 °C, and the protein concentration was determined by Bradford protein assay (Thermo Fisher Scientific). Proteins were separated by SDS-PAGE using 10% polyacrylamide gels and then transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer and immunoblotted with anti-BCL-6 (Cell Signaling, BSN, USA), anti-CD40L (Abcam, Cambridge, UK), anti-ICOS (Abcam), and anti-GAPDH (Cell Signaling) primary antibodies. Band intensities were quantified using Quantity One software (Bio-Rad, CA, USA).

The following Abs and reagents were used in this study: FITC anti-mouse CD3ε (#100306), FITC anti-mouse CD19 (#152404), APC anti-mouse CD21/CD35 (#123412), PerCP/Cyanine5.5 anti-mouse CD23 (#101618), and Brilliant Violet 421 anti-mouse IgD (#405725) (all from BioLegend, CA, USA); PE anti-mouse CD45 (#12-0451-82) (Invitrogen, CA, USA); anti-TCR α/β (#sc-19600), anti-CD4 (#sc-19641), and anti-GAPDH (#sc-365062) (Santa Cruz Biotechnology, TX, USA); anti-MHC Class II (#ab180779), anti-ICOS (#ab175401), anti-ICOSL (#ab138354), anti-CD40 (#ab188181), and anti-CD40L (#ab2391) (Abcam, Cambridge, UK); and FITC anti-mouse CD169 (MOMA-1, #MCA947F) (Bio-Rad, CA, USA).