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 analogue24 (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).
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 analogue24 (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).
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