Extended Data Fig. 5: RIPR-PD1 potentiates T cell expansion in tumour organoids, and RIPR activity is not strictly dependent on PD-1 blockade.
From: Immune receptor inhibition through enforced phosphatase recruitment
a, Quantification of TILs treated with intact RIPR (hRIPR-PD1(VHH)), 3C-digested control RIPR-PD1(VHH) (black) or nivolumab (nivo) in melanoma (left) or ovarian (right) patient-derived tumour organoids, at the indicated concentrations. b, Activated Jurkat T cells upregulate PD-1 (x axis) and CD69 (y axis). Anti-PD-1 staining is blocked by RIPR-PD1(VHH) (left) but is not affected by non-blocking RIPR-PD1(Cl19) (right). c, HEK293 cells were transfected with HA–PD-1 (N-term HA-tag fused to full-length PD-1) and LCK and CD45, as indicated. 48 h after transfection cells were treated with RIPR-PD1(VHH) or RIPR-PD1(Cl19) for 30 min at 37 °C at 0.5 or 1 μM, or 3C-cleaved RIPR-PD1(VHH) (1 μM) as indicated. Anti-HA magnetic beads were used for immunoprecipitation and samples were probed for anti-phosphotyrosine or anti-PD1 by western blot. Data are representative of two independent replicates. For raw source image, see Supplementary Fig. 1. d, e, Quantification of CD69 expression (d) and IFNγ secretion (e) after PBMC stimulation (as described in Fig. 3) for cells treated with 1 μM of nivolumab, RIPR-PD1(nivo) or non-blocking RIPR-PD1(Cl19). Data are mean ± s.d. from n = 3 biological replicates from 1 representative of 2 independent experiments. For representative gating strategy, see Supplementary Fig. 2. f, Jurkat T cells were activated overnight with plate-bound OKT3 at 0.1 μg ml−1 and incubated with 1 μM of RIPR-PD1(VHH) (blocking) or RIPR-PD1(Cl19) (non-blocking). Data are mean from n = 2 biological replicates from 1 representative of 2 independent experiments. g, Size exclusion chromatography of mouse RIPR-PD1. h, SPR binding curves for mRIPR-PD1 (clone F2) binding to immobilized mouse PD-1. i, Resonance units measured at steady state for multiple mRIPR-PD1 concentrations tested for binding to mouse PD-1. RIPR-PD1 concentration shown ranges from 8 μM to 35 nM. Kd values were found to be around 750 nM for binding to mPD-1. In h, i, data are representative from 2 independent experiments. j, Mouse CD8+ T cells were isolated from spleen and lymph nodes of C57/B6 mice and stimulated with plate-bound anti-CD3 (2C11) antibody at the indicated concentrations plus soluble CD28 (2 mg ml−1). During activation with 2C11 and anti-CD28, cells were either left untreated or were incubated with anti-PD-1 antibody (clone RMP1-14) or mouse RIPR-PD1(RMP1-14) for 24 h. CD69 (top) and CD25 (bottom) expression was quantified by FACS. k, Pmel-1 mouse CD8 T cells were stimulated with peptide pulsed APCs and 250 nM of anti-PD1 (RMP1-14) antibody or RIPR-PD1(RMP) for 24 h. CD69 were analysed by flow cytometry. In j, k, data are mean ± s.d. from n = 2 biological replicates from 1 representative of 3 independent experiments.
