Live dead fixable violet stain

Surface staining for flow cytometry was performed by washing T cells in PBS, pH 7.4, followed by staining with LIVE/DEAD Fixable Violet Stain (ThermoFisher) at recommended concentration for 30 min on ice in the dark. T cells were then resuspended in Cell Staining Buffer (BioLegend) plus relevant antibodies (APC anti-NGFR (BioLegend)) and PE-conjugated HLA-A*02:p53 R175H tetramer (NIH Tetramer Facility) for 30 min at room temperature in the dark. Cells were washed twice in Cell Staining Buffer before resuspension for analysis. Flow cytometric analysis was performed on an IntelliCyt iQue Screener PLUS VBR (Sartorius). FlowJo v.10 was used for flow cytometry data analysis.
The gating strategy used for primary human T cells is shown in Extended Data Fig. 2. For all experiments, debris was first excluded by a morphology gate based on FSC-H and SSC-H. Nonsinglets were excluded from analysis by a single-cell gate based on FSC-H and FSC-A. Live cells were selected by gating on cells negative for staining with LIVE/DEAD Fixable Violet dye (405-nm excitation, Invitrogen). Anti-NGFR APC⁺/tetramer PE⁺ cells were gated to define successfully edited live, single T cells using appropriate compensation with single-stained controls.

Tumors were minced using a razor and digested with 1 mg/ml collagenase A and collagenase D and 0.4 mg/ml DNase I in PBS at 37 °C for 2 h with rotation at 600 rpm in a thermomixer compact (Eppendorf). 10 mM EDTA was then added to stop the enzymatic reaction. The cell suspension was passed through a 70 μm filter and stained with live/dead fixable violet stain (Thermofisher Scientific). Cells were subsequently stained with the following fluorescently conjugated antibodies; CD45 (30-F11), Ly6G (1A8), F4/80 (BM8), CD11b (M/170), CD11c (N418), Thy1 (30-H12), Podoplanin (8.1.1.), PDGFRα (APA5; all from Biolegend) and CD31 (390; eBioscience) at 1:300 dilution. Flow cytometry was performed on LSR Fortessa (BD Biosciences) analyzers. Unstained and single-stained compensation beads (Invitrogen) were run alongside to serve as controls. Offline analysis was carried out on FlowJo (Treestar). For in vivo PAD4 inhibitor studies, tumors were separated for flow cytometric and immunofluorescent analysis. Some GSK484 treated tumors were too small for analysis and were therefore excluded (only tumor volumes were recorded).

PBMC was thawed in phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) and stained with LIVE/DEAD™ Fixable Violet stain (ThermoFisher SCIENTIFIC, Waltham, MA, USA) for 30 min at 4 °C. Cells were washed and then stained with anti-horse antibodies or antigen at concentrations between 0.2–1 μg/mL. After washing again, cells were resuspended in 2 mL PBS (2% BSA) and sorted as IgG+ and antigen single or double positive, at one cell per well, into PCR plates containing Rnasin^® Plus Ribonuclease Inhibitor (Promega, Madison, WI, USA) and IGEPAL CA-630. At completion, PCR plates were flash frozen in liquid nitrogen and stored at −80 °C.

Non-human primate (NHP) peripheral blood mononuclear cells (PBMCs) were stained with LIVE/DEAD Fixable Violet Stain (ThermoFisher, Waltham, MA) for 10 min at room temperature. Cells were washed with Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) with 4% fetal bovine serum (FBS) and incubated with CD3-BV421 (clone SP34–2, BD Biosciences), CD4-BV421 (clone OKT4, BioLegend), CD8-BV421 (clone RPA-T8, BioLegend), CD14-BV421 (clone M5E2, BioLegend), CD20-Cy5.5PerCP (clone 2H7, BioLegend), IgG-A700 (clone G18–145, BD Biosciences), IgM-FITC (clone G20–127, BD Biosciences), FP9-PE, and FP9-APC-Cy7 for 20 min at room temperature. The two FP probes, FP9-PE and FP9-APC-Cy7, were made by conjugating biotinylated nine amino acid HIV-1 fusion peptide (AVGIGAVF) to fluorescence labeled streptavidin and used for staining FP-specific cells. Cells were then washed two times with RPMI with 4% FBS and assessed by flow cytometry. FP double-positive IgG+ memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20+IgG+IgM-FP9+) were sorted into 384-well culture plates at single-cell per well using a FACSAria II sorter (BD Biosciences), and relevant index information recorded. We carefully recorded all cell events during sorting and used the exact numbers of events from the sorter to calculate percentage of probe-stained cells in total IgG+ B cells.