Fig. 2: Probe H5 detects GzmB-mediated anticancer activity of CD8+ T cells. | Nature Communications

Fig. 2: Probe H5 detects GzmB-mediated anticancer activity of CD8+ T cells.

From: A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies

Fig. 2

a Schematic procedure for co-culture assays. b Representative microscopy images of CD8+ T cells before/after reinvigoration and staining by anti-GzmB (10 nM, red) and Hoechst 33342 (1 µM, blue) (n = 3). Scale bar: 10 μm. c Flow cytometry (gating: Fig. S7) of E0771 cells in co-culture with active (IL-2, 24 h) or inactive (IL-2, 2 h) CD8+ T cells, or alone with staurosporine (1 μM, 1 h). Legends: viable (gray), H5-stained (green), apoptotic (red). H5 (5 μM), Annexin V-AF647 (10 nM). Data as means ± SEM (n = 3). d Confocal microscopy of mKate-expressing E0771 cancer cells (red) stained with H5 (25 μM, green) in co-culture with active T cells (left), non-active T cells (centre), or active T cells plus Ac-IEPD-CHO (right). Black arrows highlight T cells, yellow arrows highlight H5-stained intracellular GzmB puncta. Quantification of fluorescence intensity by image analysis shown in Fig. S8. Scale bar: 10 μm. e Time-course fluorescence microscopy of OT-I CD8+ T cells stained with Cell Tracker Orange (red) killing OVA-EL4 cancer cells in the presence of H5 (10 μM, green) and Sytox Blue (1 μM, blue) (n = 3). Scale bar: 10 μm. f Flow cytometric analysis of OVA-EL4 cancer cells from experiments in e. Data as means ± SEM (n = 3). P-values from two-tailed t-tests. Source data (c, f) provided as a Source Data file.

Back to article page