Fig. 5

Direct effects of AZD8835 (PI3Kα/δ) and CAL-101 (PI3Kδ) inhibitors in CD8+ T-cell activation ex vivo via IL-2 autocrine loop. Naïve CD8⁺ T-cells were purified from spleen, labelled with CTV and stimulated for 3d with α-CD3/α-CD28 coated activator beads. Inhibitors were added at indicated concentrations. a Histogram shows representative proliferation as measured by CTV dilution following culture. Line graph shows proliferation index. b Representative flow cytometry plots of T-cell viability in the presence of AZD8835 and CAL-101. Line graph shows viability quantification related to vehicle control. c Purified naïve CD8⁺ T-cells were left unstimulated (black line) or stimulated for 3 days in the presence of 1 μg/mL αCD3 and 1 μg/mL soluble CD28 (grey fill), stimulated for 3 days with a 1:1 ratio of Dynabeads mouse T-cell activator with AZD8835 (blue line) or CAL-101 (green line). Representative histograms of purified CD8+ T-cells stimulated for 3d with a 1:1 ratio of Dynabeads mouse T-cell activator. Histograms show cell size (c) expression of (d) CD69 and (e) CD25 on indicated populations. Data represents 2 experiments. f Ex vivo protein secretion of IL-2 by CD8+ T-cells in a dose range of AZD8835 and CAL-101. g Purified CD8⁺ T-cells were stimulated in the presence of increasing concentrations of AZD8835 or vehicle +/− 10 μg/mL αIL-2 neutralizing antibody. Cell viability of purified naïve CD8⁺ T-cells stimulated as in (C) with vehicle or 10 ng/mL of recombinant murine IL-2 in the presence of inhibitors at 0.3 μM concentration. Statistical significance is indicated as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data representative of > 2 independent experiments