Anti icos c398.4a

To analyze lymphoid populations within the tumor from one patient (patient 4), the fresh tumor tissue was finely minced with scissors and filtered through a nylon membrane, and a single-cell suspension was purified over a Ficoll–Hypaque density gradient. Cells were stained with the following anti-human antibodies: anti-αβTCR (IP26, eBioscience, San Diego, CA, USA), anti-CD8 (OKT8, eBioscience), anti-CD44 (IM7, eBioscience), anti-PD-1 (eBioJ105, eBioscience), anti-CD27 (O323, eBioscience), anti-CD20 (2H7, eBioscience), anti-CD1c (L161, eBioscience), anti-γδTCR (B1, BioLegend, San Diego, CA, USA), anti-CD4 (OKT4, BioLegend), anti-CD28 (CD28.2, BioLegend), anti-FAS (DX2, BioLegend), anti-ICOS (C398.4A, BioLegend), anti-IgM (MHM-88, BioLegend), anti-CD21 (B-ly4, BD Biosciences, San Jose, CA, USA), anti-CD23 (M-L233, BD Biosciences), and anti-IgD (Southern Biotech, Birmingham, AL, USA). Flow cytometry was performed using a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed via FlowJo software (Tree Star, Ashland, OR, USA).

For flow cytometry cell sorting, cells were stained with anti-CD4 (RPA-T4) and anti-CD3 (SK7) antibodies (BioLegend); anti-CD4 (RPA-T4), anti-CD3 (SK7) and anti-HLA-DR (L243) or with anti-CD4 (SK3), anti-CD3 (UCHT1), and anti-CD14 (63D3) according to the sorting strategy. Gating strategies are depicted in Supplementary Figs 1–3. Sorted populations cell purity was routinely >98%. For intracellular cytokines assays sorted CD3^highCD4^high, rested for at least 3 h, were stimulated with 5 μg mL⁻¹ of anti-CD3 (UCHT1, BioLegend) and 2 μg mL⁻¹ of anti-ICOS (C398.4 A, BioLegend), crosslinked with 5 μg/mL anti-mouse IgG1 (BioLegend) plus 10 μg mL⁻¹ anti-hamster IgG (Thermo Fisher Scientific) at 37°C in the presence of Brefeldin-A (Life Technologies) for 14 h. Cells were fixed in paraformaldehyde 1% (Sigma-Aldrich) and permeabilized with saponin (Carl Roth). Antibodies used are listed in Supplementary Data 2. When indicated 1.7 μg mL⁻¹ LPS (Sigma-Aldrich), TNC (Merck Millipore), or cell-depleted synovial fluid (SF) was added. For TLR4 blocking, CLI-095 (InvivoGen) was added at 10 μg mL⁻¹ 1 h before stimulation. Cell sorting was performed in a BD FACSAria III instrument (BD Biosciences).