Fig. 4. Probe H5 detects immunomodulatory action in phenotypic screens and T cell cytotoxic activity in human tissue from lung cancer patients.

a Experimental protocol of the phenotypic screen. b Fluorescence intensity of probe H5 in co-cultures of E0771 cells and IL-2-activated CD8+ T cells after incubation with small molecules (C1-C44). Wells received 100 U mL⁻¹ IL-2 (low IL-2) for T cell viability. High IL-2 (250 U mL⁻¹) used as a positive control for invigorated CD8+ T cells and rapamycin (0.3 μM) used as a negative control. Probe H5 (20 μM) and Hoechst 33342 (1 µM) were incubated for 1 h and fluorescence images were acquired with an ImageXpress^TM XLS. Data as means ± SEM (n = 8). Chemical structures of drugs showing H5 fluorescence signals above those with 250 U mL⁻¹ IL-2. c Representative H&E microscope images of non-tumor (left) and lung adenocarcinoma (right) paired samples. Scale bar: 50 µm. d Cytometry analysis (gating: Fig. S17) showing the percentage of EpCAM+ H5+ epithelial cells found in paired tissues (non-tumor vs tumor) from lung cancer patients after incubation with probe H5 (5 μM). Data as means ± SD (n = 5). P-value from two-tailed t-tests. Source data (b, d) provided as a Source Data file.