Figure 6. Glutamine antagonism increases immunogenic cell death and antigen presentation to T cells.

(A–C) 4T1 tumor cells were cultured with or without DON (0, 0.5, 1, 5, 10, or 50 μM) for 24 hours. (A) Cells were harvested and stained with CellROX (ROS measurement) and analyzed by flow cytometry. Representative histogram (left) and summary graph (right). (B) Cells were lysed and active caspase-3 was analyzed by immunoblot. (C) Cells were stained for calreticulin and analyzed by flow cytometry. Percentages of surface calreticulin⁺ in vitro 4T1 cells are shown (left). Summary graph of surface calreticulin gMFI on GFP⁺ gated in vivo tumor cells (right). (D) 3LL cells were cultured with or without DON (0.5 or 1 μM). After 1 hour of incubation, cells were washed and replaced with drug-free media. After 24 hours, supernatants were harvested and used as conditioned media (CM). BMDMs were cultured in the presence of these conditioned media for 24 hours. Phospho-NF-κB (Ser536) and LAMP2 were measured by immunoblot. (E–G) BMDMs (3 × 10⁵) and 5 × 10⁴ B16-OVA tumor cells were cocultured with or without DON (1 or 5 μM). After 24 hours of incubation, supernatants were discarded and 3 × 10⁵ eFluor 450–labeled naive CD8⁺ T cells from OTI mice were added. (E) Schematic of the experiment. (F) Representative flow plot (left) and percentages of divided cells (right) from CD8⁺ T cells were analyzed by flow cytometry. (G) BMDMs from WT, MyD88/TRIF double KO (DKO), or TFEB-KO mice and B16-OVA tumor cells were cocultured using the same method as in E, and a histogram of the dilution of eFluor 450–labeled CD8⁺ T cells is shown. Data are representative of at least 3 independent experiments and are presented as the mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparisons post hoc test (A; C, left; and F) or Mann-Whitney test (C, right).