Extended Data Fig. 1 |. Tonic PD-1 signalling dampens T cell activation.

a, TCR downregulation in wild-type or transduced Jurkat T cells with CRISPR/Cas9 and gRNA targeting PD-1 (J-CRISPR-PD-1) upon OKT3 stimulation overnight. b, Jurkat T cells transduced with wild-type human PD-1 (Jurkat-PD1). c, PD-1 versus CD69 expression for Jurkat T cells stimulated overnight with OKT3. Data are shown as individual biological replicates, n = 3. d, CD69 upregulation after OKT3 and anti-CD28 stimulation overnight of wild-type Jurkat cells or Jurkat-PD1 cells (shown in b) or PD-1 KO cells (J-CRISPR-PD-1). In a, d, data are shown as mean ± s.d., n = 3 biological replicates from 1 representative of 3 independent experiments. e, f, Jurkat cells were transduced with control gRNA/CRISPR plasmid or gRNA targeting the endogenous PD-1 gene and activated at increasing concentrations of plate-bound OKT3 overnight. In a separate control group, Jurkat cells were treated with anti-PD1 (Nivolumab), anti-PD-L1 and anti-PD-L2 antibodies (50 μg ml⁻¹ for each antibody). CD69 (e) and PD-1 (f) expression was quantified by flow cytometry. Data are mean ± s.d. from n = 2 biological replicates from 1 representative of 2 independent experiments. g, Expression of HA–PD1-ICD (ECD-less) reduced the upregulation of CD69 12 h after activation with plate-bound OKT3 at the indicated concentrations. Data are mean ± s.d. from n = 3 biological replicates from 1 representative of 2 independent experiments. h, i, Quantification of PD-L1 and PD-L2 in resting and activated Jurkat cells. Resting or activated Jurkat T cells, were incubated with anti-PD-PL1 (h), anti-PD-L2 (i) or with appropriate isotype controls and analysed by flow cytometry. Expression of PD-L1 and PD-L2 was not detected in either resting or activated Jurkat T cells. Non-fluorescent anti-PD-L1 or L2 antibody (right) was added before labelling as a ‘blocking’ control. In h, i, data are representative of 3 independent experiments.