Extended Data Fig. 6: In vitro testing of mouse RIPR-PD1.
From: Immune receptor inhibition through enforced phosphatase recruitment
a–e, CD4+ T cells were stimulated for 3 days in the presence of anti-CD3/CD28 and anti-PD1, anti-PD-L1(VHH) or RIPR-PD1, all at 1 μM, after which cells and supernatant were collected by analysis for proliferation (a), IL-2 (b) or IFNγ (c) secretion, fraction of CD44highCD62Llow (d) and CD25 (e). Cell proliferation was quantified using CellTiter-Glo, surface expression by flow cytometry and cytokine secretion by ELISA. f, Mouse CD4+ T cells were stimulated with plate-bound anti-CD3 and soluble CD28 in the presence of absence of plate-bound PD-L1 or isotype control. Cells were treated with anti-PD1 antibody (RMP1-14) or RIPR-PD1 at 1 μM and supernatant was collected for quantification of IFNγ production on day 3. In a–f, data are mean ± s.d. from n = 3 biological replicates from 1 representative of 2 independent experiments. g, Size-exclusion chromatography of intact or 3C-cleaved mouse RIPR-PD1. h, Coomassie-stained SDS–PAGE of mouse mRIPR-PD1 uncleaved or treated with 3C overnight. i, Mouse CD8+ T cells were activated with plate-bound 2C11 (1 mg ml−1) and soluble CD28 (2 mg ml−1) for 24 h and treated with 1 μM of intact (middle) or 3C-treated (right) mRIPR-PD1. j, k, Quantification of the CD69 (j) and CD25 (k) expression of representative data shown in i. Data are mean from n = 2 biological replicates from 1 representative of 3 independent experiments. l, Competition experiment between mRIPR-PD1 at the indicated concentrations and anti-CD45-MSA (2 μM), anti-PD-1-MSA (2 μM) or both, as indicated. Mouse CD8+ T cells were stimulated with 8 μg ml−1 of 2C11 and 2 μg ml−1 of anti-CD28. Black line indicates CD69 expression (with 2C11 and anti-CD28 only). CD69 expression was quantified by FACS 24 h post-stimulation. Data are mean from n = 2 biological replicates from 1 representative of 3 independent experiments. m, n, Competition experiment between mRIPR-PD1 and soluble mouse PD-1 (5 μM). Mouse CD8+ T cells were stimulated with 2 μg ml−1 (m) or 4 μg ml−1 (n) of plate-bound 2C11 and 2 μg ml−1 of soluble anti-CD28. CD69 was quantified by FACS 24 h after stimulation. Data are mean from n = 2 biological replicates from 1 representative of 3 independent experiments.
