Extended Data Fig. 5: Anti-PD-L1 antibody target engagement within the peripheral immune system.

(a) Flow cytometry plots demonstrating the gating strategy for T cells (CD3⁺) from the blood of WT mice. (b) Representative histogram plots of anti-rat IgG2b FITC fluorescent intensity, as a measurement for PD-L1 occupancy, in the saturated tube (‘sat’, orange) and in the tested tube (‘test’, purple) (see ‘Methods’) on CD3⁺ T cells in blood samples from WT mice, 7 days following treatment with anti-PD-L1 antibody in dose of 0.1, 0.5 or 1.5 mg/mouse, or with 1.5 mg/mouse of IgG2, or untreated (n/group=6; 3 males, 3 females). (c,d) Quantification of the percentage of PD-L1 receptor occupancy (%RO) on T cells (mean ± SEM) from the blood (c) and spleen (d). The %RO is calculated as geometric mean of ‘test’ divided by geometric mean of ‘sat’. (e) Flow cytometry plots demonstrating the gating strategy for PD-1⁺ effector memory T cells (T_EM, CD4⁺CD44⁺), measured in the blood of the same mice as detailed above. (f) Percentage of PD-1⁺ T_EM cell out of total T_EM (Mean ± SEM). One sample from the group of anti-PD-L1 1.5 mg/mouse was not included for technical reason. (c,d,f) were analyzed using one-way ANOVAs ([c] F_(4,25)=7.23, p=0.0005, R²=0.536; [d] F_(4,25)=7.16, p=0.0005, R²=0.534; [f] F_(4,24)=10.71, p < 0.0001, R²=0.641) followed by Fisher’s LSD tests (n.s. – not significant, ** p < 0.01, *** p < 0.0001). data in all graphs are shown as mean ± SEM.