Fig. 5: NOTCH1ΔPEST up-regulates PD-L1 on CLL cells.
a Heatmap of the top 50 differentially expressed proteins identified by mass spectrometry. Histogram with the Log2FC values from three independent patients analyzed is shown. b PD-L1 expression analyzed on GFP-positive cells 5 days post transduction and quantified as ratio of MFI of CLL (n = 20) and cell lines (n = 3 repeats) transduced with NOTCH1ΔPEST or an empty vector control. c Quantification of PD-L1 on NOTCH1-mutated CLL cells treated for 48 h with a γ-secretase inhibitor (10 µM) or control (DMSO) (n = 4; p = 0.032). d PD-L1 expression quantified as ratio of MFI for CLL cell transduced with an empty vector (blue) or NOTCH1ΔPEST (red) during cell cycle progression (n = 4). Cells were pulsed for 12 h with Edu (p = 0.017, 0.03 and 0.01, respectively). e Comparison of PD-L1 expression on CLL cells, transduced with NOTCH1ΔPEST or c-MYC relative to the empty vector control (n = 3; p = 0.037). f Bar graph of the Log2FC values of IFNG and IFNGR1 expression analyzed by RNAseq following NOTCH1ΔPEST transduction (n = 13). g PD-L1 expression of CLL cells (n = 7) transduced with an empty vector or NOTCH1ΔPEST and treated with a blocking IFNG receptor antibody for 24 h; (p = 0.011). h. Schematic diagram of the experimental setup for data shown in panel j. i CD19 expression on CLL cells transduced with an empty vector (gray) or NOTCH1ΔPEST (orange) 5 days post transduction (n = 5), assessed by flow cytometry. Representative flow cytometry histogram image is shown on the left (p = 0.021). j Quantification of luciferase activity (RLU) of Jurkat NFAT-Luc reporter cells co-cultured with empty vector (gray bar), NOTCH1ΔPEST (orange bar) or NOTCH1ΔPEST CLL cells in the presence of 10 nM Blinatumomab and 0.1 nM Durvalumab (pink bar). CLL cells (n = 7) were co-cultured with Jurkat cells at a ratio of 2:5 for 24 h (p = 0.0226). Cohorts are shown as mean ± SEM. Statistical analysis was done by multiple paired t-tests (d), paired t-tests (b, c, i) or one-way ANOVA followed by paired t-tests (e, g, j). Each symbol refers to an individual patient sample. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and not significant (ns) P > 0.05.
