Figure 6. Cytokine catch-and-release could enable communication between spatio-temporally separate cells and is also observed for IL12.

See also figure S6. (A) Diagram of experiment. B16 IFNγR KO cells were pulsed with IFNγ, washed, and then co-cultured with either wildtype or IFNγR KO BMDM. (B) Macrophage activation was assessed by iNOS expression by flow cytometry. Data are representative of at least 3 independent experiments. (C) Schematic of experiment. B16 cells were stimulated with IFNγ for 5 hours and then washed. After washing, one cohort of cells received TNFα and another received TNFα and αIFNγ. (D) Cell viability was assessed by DAPI incorporation. Data are representative of 2 independent experiments. (E) Catch-and-release communication could enable activated T cells to release IFNγ, even after IFNγ production has been shut down. (F) Naïve or activated T cells were split into three cohorts where cohort 1 was left untreated, cohort 2 was pulsed with IFNγ for 4 hours, and cohort 3 was treated with a combination of drug inhibitors to abrogate IFNγ production. All cohorts were then co-cultured with B16 sensor cells and sensor MHC-I (H2-K^b) up-regulation was quantified by flow cytometry after 1 day. Data are representative of 3 independent experiments. (G) Lipid-spotted strips were incubated with 5nM IL12, probed with antibodies directed against IL12, and developed. Blot is representative of 3 independent experiments. (H) IFNγR KO B16 cells were pre-treated with Annexin V, or Annexin V binding buffer before performing the IL12 cell capture assay. Data are representative of at least 3 independent experiments. (I) B16 IFNγR KO cells were pulsed with 10nM IL12, then washed and co-cultured with BALB/c wildtype BMDM. Production of IFNγ was quantified by bead-based ELISA. Data are representative of 3 independent experiments. N.D. stands for not detected. (J) Thyroid tumors or healthy thyroids were isolated and dissociated into single cell suspensions and cultured for 1h with a combination of drug inhibitors to abrogate IFNγ production. Tumors or healthy thyroids were then co-cultured with labeled B16 (IFNγR+/+ or −/−) for 48 hours. MHC-I was quantified by flow cytometry across live DAPI negative cells. n of tumor-bearing and healthy thyroids = 3 each. The cytokine sharing assay was repeated three independent times.