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. Author manuscript; available in PMC: 2021 Nov 18.
Published in final edited form as: Cancer Immunol Res. 2020 Nov 11;9(1):34–49. doi: 10.1158/2326-6066.CIR-20-0080

Figure 2.

Figure 2.

The IFNγ signaling pathway is a central regulator of cancer cell resistance. A, MOLM13, SEM, and HCC827 cells were treated with a blocking antibody against IFNGR1 (α-IFNGR) and applied in the RTCC assay with corresponding CD3-bispecific antibodies (n = 6). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. B–D, MOLM13, SEM, and HCC827 cells were knocked out for JAK1 (JAK1KO) and STAT1 (STAT1KO), and killing by CD3-bispecific antibodies was compared with control groups (Scramble; n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. E, Scramble and PTPN2KO cells were starved in RPMI1640 overnight and treated with IFNγ for 5 minutes. IFNγ signaling was reported by STAT1 phosphorylation (pSTAT1) in Scramble and PTPN2KO MOLM13 cells. F, RTCC assay of PTPN2KO MOLM13 cells (n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 24 hours after treatment. G, The growth of MOLM13 (n = 3), SEM (n = 5), and HCC827 (n = 8) cells 72 hours after IFNγ treatment. The red dashed line indicates the maximum IFNγ release in the corresponding RTCC assay (referring to Supplementary Fig. S2D). H, Heatmap of the overlapped hits from the IFNγ treatment RNA-seq and CRISPR screen datasets of each individual cell line. I, Flow cytometric analysis of ICAM1 and FAS expression in the MOLM13 and SEM cells with IFNγ titration (n = 3). J, Western blot of caspase family protein expression in the three cell lines 24 hours after IFNγ (100 ng/mL) treatment (n = 1). K, RTCC assay of ICAM1KO MOLM13 cells using the CD123-DART or CD33-BiTE (n = 6). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 24 hours after treatment. bsAb, bispecific antibody. L, RTCC assay of FASKO SEM cells using the CD123-DART or CD19-BiTE (n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. M, RTCC assay of CASP8KO HCC827 cells using the PCAD-DART (n = 4). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 48 hours after treatment. For each plot, error bars represent SEM.