Phosphoflow perm buffer 3

Cells were surface stained for surface markers using respective antibodies and cell staining buffer (Biolegend). For intracellular phosphorylated (p)- Zap70 (pZap70) and Akt (pAkt), cells were fixed with Fixation Buffer and permeabilized with Phosphoflow Perm Buffer III (both from BD Biosciences) followed by staining with PE–anti-pZap70^(pY292) or –anti-pAkt^(pS473). PBMCs were stained for intracellular FoxP3, IFNγ and IL10 using Cytofix/Cytoperm Plus kit and anti-FoxP3, -IFNγ and -IL10 (all from eBioscience). Flow cytometry was done on FACS Canto and data analyzed using Flowjo (Treestar). Controls for each experiment included unstained cells, fluorescence minus one (FMO) and isotype matched antibodies.

After 3 d of in vitro activation, cells were collected and washed multiple times to remove endogenous IL-2. A portion of the CFSE-labelled T cells were exposed to 10 µg ml^–1 of parental anti-PD-1 antibody to block the PD-1 epitope for 30 min at room temperature and, thereafter, unbound antibody was washed away.
To assess IL-2R signalling (STAT5-P) on human T cells following treatment, both anti-PD-1-pretreated and untreated cells were exposed to increasing concentrations of PD1-IL2v, FAP-IL2v or FAP-IL2 superkine analogue^{24 (link)} for 12 min at 37 °C. To investigate the cis/trans binding of PD1-IL2v, anti-PD-1-pretreated or untreated CFSE-labelled cells were co-cultured 1:1 with untreated CTV-labelled cells. Cells were then exposed for 12 min at 37 °C to 0.1 μg ml^–1 (630 pM) of the treatment fusion proteins.
For the mouse ex vivo experiment, the cells were treated with increasing doses of muPD1-IL2v or muFAP-IL2v for 30 min at 37 °C.
Directly after treatment, cells were fixed with Phosphoflow Fix Buffer I (BD) and incubated for 30 min at 37 °C. Cells were then permeabilized overnight at −80 °C with Phosphoflow PermBuffer III (BD) before being stained for 30 min at 4 °C with anti-STAT5-P–AF647 antibody (clone 47/pY694, BD Biosciences; 1:20).

Starved and sensitized BMMCs [WT and SLAP KO (20 × 10⁶ cells)] were washed twice in PBS and then re-suspended in RPMI (Phenol red free) supplemented with 10 mM HEPES 7.4, 1 mM sodium pyruvate and 1% (v/v) minimum non-essential amino acids for 4 h. Subsequently, cells were stimulated in Tyrode’s buffer with vehicle or DNP-HSA (30 ng/ml) for indicated time-points and fixed with 2% paraformaldehyde for 10 min at 37°C, followed by washing with PB (PBS supplemented with 1% bovine serum albumin) buffer twice before permeabilization with cold (−20°C) BD phosphoflow perm buffer III for 30 min. After permeabilization, cells were stained with antibody solution (1:100 dilution) for 30 min at room temperature before analysis on a cytometer. The following BD Bioscience antibodies for phosphoflow analysis were used: pSyk (pY348, PE; clone 1120-722) and pNFkB p65 (S529, PE; clone K10-895.12.5).

0.5 million PBMCs were stimulated with 10 or 20 ng/mL of recombinant human IL7 (peprotech cat # 200–07). Cells were subsequently fixed with 1 × Lyse/Fix (BD Biosciences #558,049) and permeabilized with BD phosphoflow Perm Buffer III (BD biosciences #558,050) and stained with BV786 anti-human CD3 (BD biosciences #563,800), V450 anti-human CD4 (BD biosciences #561,838), APC-Cy7 anti-human CD8 (Biolegend# 344,714) and PE anti-pSTAT5 (BD biosciences #612,567). Stained cells were acquired on NovoCyte flow cytometer.