Anti cd45ra clone hi100

Fresh tumor tissue was obtained for CITE-seq. Tumor cells were dissociated, washed, and resuspended. Antibodies for CITE-seq were anti-CD34 (clone 581), anti-CD31 (clone WM59), anti-CD45RA (clone HI100), anti-CD3 (clone UCHT1), anti-CD8A (clone RPAT8), anti-CD4 (clone RPAT4), anti-CD14 (clone 63D3), and anti-CD19 (clone HIB19) TotalSeq-A antibodies (BioLegend, San Diego, CA). Cells from one tumor were used to optimize preparation by adding variable antibody concentrations to 1 million cells in 50 mcl, washing with 200 mcl FACS buffer, resuspending in 200 mcl FACS buffer, and analyzing by flow cytometry. Otherwise, cells were used for CITE-seq using 3′ v3 Single Cell Immune Profiling technology according to the manufacturer’s protocol (10× Genomics, San Diego, CA).

All clinical flow cytometry (FACS) was performed following good clinical practice at the Department of Clinical Immunology, Addenbrooke’s Hospital, Cambridge, UK, within 4 h of phlebotomy. Operators were blinded to the aldesleukin dose allocated. A 2.6-ml sample of peripheral whole blood was collected into EDTA tubes, and 50 μl was stained with specific fluorochrome-conjugated antibodies at room temperature for 15 min to identify Tregs as CD3⁺CD4⁺CD25^highCD127^low T cells. The clones used were anti-CD3 (clone SK7, phycoerythrin [PE]-Cy7-labelled; BD Biosciences), anti-CD4 (clone RPA-T4, FITC-labelled; BD Biosciences), anti-CD127 (clone HIL-7R-M21, PE-labelled; BD Biosciences), anti-CD25 (clone M-A251 and 2A3, allophycocyanin [APC]-labelled; BD Biosciences), anti-CD45RA (clone HI100, APC-Cy7-labelled; BioLegend), and anti-CD62L (clone DREG-56, PerCP/Cy5.5-labelled; BioLegend). Red cells were then lysed (BD FACS Lysing Solution); the cells were washed and resuspended in BD Cell Fix and then immediately analysed on a BD FACSCanto II flow cytometer utilising FACSDiva software (BD Biosciences). In parallel, a whole-blood BD Multitest 6-Color TBNK assay using BD Trucount Tubes according to the manufacturers’ instructions (BD Biosciences) was run to determine the relative and absolute concentration of lymphocyte subpopulations, including T, B, and NK cells.