Lymphoprep density gradient separation

Tumors were dissociated into a single cell suspension using the Tumor Dissociation Kit and gentleMACS Octo Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. TILs were separated using the Lymphoprep density gradient separation (STEMCELL), and the human T cell populations were analyzed by flow cytometry based on the surface expression of human CD45, CD3, CD4, CD8, and Foxp3. Antibodies used for TILs analysis were purchased from Biolegend. TILs populations were determined using a Attune™ NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software.

Human PBMCs were isolated from healthy volunteers using the Lymphoprep density gradient separation (STEMCELL, #07811). To improve the sensitivity of T cells to immunostimulation, isolated PBMCs with high cell density were pre-cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gioco). Subsequently, pre-cultured PBMC were stimulated with soluble or immobilized isotype IgG (anti-hen egg lysozyme antibody, AP Biosciences), anti-CD3 antibody (OKT3, Biolegend), anti-CD28 antibody (TGN1412, ichorbio), and bispecific antibody AP203 for 24 h. For immobilized antibodies, the antibodies were coated in the Maxisorp plate overnight at 4 °C. The secreted cytokines in culture supernatant were determined by ProcartaPlex Immunoassays (Thermo Fisher Scientific) was used to measure the secreted cytokine in culture supernatant, including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-13, IL-17A, TNF-α, and IFN-γ.