Fig. 1.

The inhibitory function of PD-1 requires PD-Ligands to bear mechanical support. (A) Schematics of stimulating NFκB::eGFP reporter Jurkat cells with T-cell stimulator cells (TSC) expressing a single-chain fragment variable (scFv) of anti-CD3 (clone OKT3) and PD-L1 or PD-L2. (B) Representative SSC vs GFP plots of reporter Jurkat cells 24 hr after stimulation with indicated conditions. (C-D) Quantification of GFP expression for conditions in B. n = 5 – 6 for plain pooled from 3 independent experiments or n = 9 – 10 for PD-1 reporter cells pooled from 5 independent experiments. (E-F) Schematics of stimulating NFκB::eGFP reporter Jurkat cells with soluble anti-CD3 and soluble (E) or bead-coated (F) PD-Ligands. (G-H) Quantification of GFP expression for conditions in E&F. n = 4 for all conditions pooled from two independent experiments. (I) Schematics of stimulating NFκB::eGFP reporter Jurkat cells with soluble anti-CD3 and PD-Ligands coated beads without or with [PEG]₂₄ spacer arm. (J-K) Quantification of GFP expression for conditions in I. n = 10, 10, and 8 for SA, PD-L1, and PD-L2, respectively, pooled from 5 independent experiments. Normalized frequency (C, G, and J) and normalized geometric mean fluorescence intensity (gMFI) (D, H, and K) were calculated as (sample – averaged background)/(anti-CD3 control – averaged background). Numbers on graphs represent p values calculated from two-tailed student t-test.