Anti ctla 4

T cell culture was performed in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (LabTech), 2 mM L-Glutamine (Sigma-Aldrich), 1% penicillin and streptomycin (Invitrogen) at 37°C, 95% humidity, 5% CO2. Assessment of memory T cell proliferation and phenotype was undertaken following incubation with 5μM Cell Trace Violet (ThermoFisher Scientific) in PBS for 20 min at 37°C, then washing 3 times in media. Where indicated, the following antibodies and cytokines were added: 2 μg/ml anti-CD80 (R&D systems), 2 μg/ml anti-CD86 (R&D systems), 20 μg/ml anti-CTLA-4 (Ticilimumab) (a gift from Pfizer), 1 μg/ml anti-CD28, recombinant human Interleukin 2 (PeproTech).

Cells were incubated at 37°C with unlabelled anti‐CTLA‐4 (ticilimumab, a gift from Pfizer) in media for 1 h to tag surface and internalized CTLA‐4, and then washed with ice‐cold media and placed on ice. Antibody‐bound surface CTLA‐4 was stained with Goat anti‐Human IgG‐Alexa Fluor 647 (Thermo) in media on ice for 10 min in the dark. Cells were either washed and analysed for surface stain to provide a baseline or transferred to 37°C to allow trafficking to resume in the presence of Goat anti‐Human IgG‐Alexa Fluor 647. Recycling of antibody‐bound CTLA‐4 was detected as the increase in Alexa Fluor 647 at the time‐points shown. In LRBA KO Jurkat cells, after the 37°C recycling stain, cells were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.1% saponin in media. The cells were then stained for total CTLA‐4 (F‐8 PE, Santa Cruz) and with rabbit anti‐LRBA for 30 min, followed by Donkey anti‐Rabbit IgG‐Alexa Fluor 488.