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. 2024 Jan 16;221(2):e20230488. doi: 10.1084/jem.20230488

Figure 6.

Figure 6.

Human CD8 T effector cells can support tumor spheroid growth. (A) CD8 Tnaive, Tcm, and PD1+TIGIT+ were isolated from human blood and ex vivo cultivated (resting) or ex vivo cultivated and activated (stimulated). Cell-free SNs were used in a spheroid assay, and spheroid growth was observed over time. Left, representative images with bead control, unstimulated, or stimulated Tnaive, Tcm, or PD1+TIGIT+ CD8 T cells. Right, largest object areas over cultivation time with statistical verification across experiments using normalized AUC (n = 4–6, one-way ANOVA). Scale bars = 400 µm; enhanced for improved visibility. (B) Spheroid assay using cell-free SN from influenza-specific CD8 T cells and varying amounts of pulsed peptides on MRC-5 and HaCaT cells seeded in a 30:70 ratio with statistical verification across experiments using normalized AUC (n = 4, one-way ANOVA of AUC, symbols indicate individual experiments), experimental repeat in Fig. S4. Scale bars = 400 µm; enhanced for improved visibility. (C) Spheroid assay using cell-free SN from TIL-target cell coculture with statistical verification across experiments using background-subtracted AUC (n = 10, one-way ANOVA of AUC, symbols indicate individual experiments), experimental repeat in Fig. S4. Scale bars = 400 µm; enhanced for improved visibility. (D) Expression of CD39 and PD1 in CD8 T cells from tumor patient blood, liver tumor, liver NAT, fat, and skin; statistics to the right (n = 3–7, one-way ANOVA). (E) Intracellular deposition of IFN-γ and TNF in PMA/ionomycin and TI-stimulated liver tumor, liver NAT, and fat CD8 T cell subpopulations (n = 4–17, one-way ANOVA). (F) Spheroid assay using recombinant cytokines. Left, representative images with carrier, IL-10, TNF, and IFN-γ. Right, largest object area over cultivation time, with statistical verification across experiments using background-subtracted AUC (n = 3, one-way ANOVA of AUC). Scale bars = 400 µm; enhanced for improved visibility. All data are derived from two or more independent experiments with the indicated number of replicates.