Figure 5.
TNFα signals through JAK1 to upregulate ICAM-1. (A) KG-1a and OCI-AML2 cells were exposed to increasing or fixed (40 μM) concentrations of RIPK1 inhibitor, JAK1 inhibitor, or dimethyl sulfoxide (DMSO) for 24 hours, followed by rTNFα treatment. AML were then cocultured with DNTs for 24 hours (for KG-1a) or 2 hours (for OCI-AML2). Percentage increase in specific killing after rTNFα pretreatment was determined. The data shown are representative of 2 independent experiments. (B-C) WT OCI-AML2 and OCI-AML3 cells were untreated or transduced with shRNAs against JAK1 (shJAK1) or GFP (shGFP). RNA expression of JAK1 was normalized to HPRT housekeeping gene (B). Transduced AML cells were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs for 2 hours. Specific killing (left) and percentage increase in specific killing (right) after rTNFα pretreatment are shown (C). (D) AML cell lines (top), primary AML samples with patient IDs, and healthy PBMCs (bottom) were untreated (blue) or treated (orange) with rTNFα. Representative histograms of ICAM-1 expression with MFI values including fluorescence-minus-one (FMO) control (red) are shown. (E) CD33+CD45lowCD3−CD34+ (CD34+) and CD33+CD45lowCD3−CD34− (CD34−) primary AML blasts from 2 patients were treated with rTNFα. The ICAM-1 MFI fold change is shown. (F) ICAM-1 expression with MFI values of AML cell lines cocultured with or without DNTs for 24 hours in the presence of anti-TNFα or isotype control. The data shown are representative of 2 independent experiments. (G) MV4-11 and KG-1a were cocultured with or without DNTs in separate compartments of the transwell from Figure 4B. Representative histograms of ICAM-1 expression on KG-1a with MFI values after 2 days is shown. (H) KG-1a cells were alone or were incubated with MV4-11 or OCI-AML3 and DNTs in the presence of anti-TNFα blocking antibody or isotype control for 24 hours. ICAM-1 expression on live KG-1a cells was determined. The data are representative of 2 independent experiments. (I) KG-1a and OCI-AML2 were treated with JAK1 inhibitor or DMSO for 24 hours, followed by stimulation with or without rTNFα and stained for ICAM-1. The MFI fold change of ICAM-1 expression from rTNFα-treated AML cells relative to untreated AML cells is shown and compared between vehicle and JAK1 inhibitor conditions. The data displayed are 2 pooled independent experiments. (J) JAK1 knockdown (shJAK1) or control (shGFP) AML cells were untreated or treated with rTNFα (100 ηg/mL), then stained with anti–ICAM-1 antibody. The MFI fold change of ICAM-1 expression from rTNFα-treated AML cells relative to untreated AML cells is shown and compared between control and JAK1 knockdown conditions. The data shown are representative of 2 independent experiments. Student t test and 1-way/2-way ANOVA were used. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; shRNA, short hairpin RNA.

TNFα signals through JAK1 to upregulate ICAM-1. (A) KG-1a and OCI-AML2 cells were exposed to increasing or fixed (40 μM) concentrations of RIPK1 inhibitor, JAK1 inhibitor, or dimethyl sulfoxide (DMSO) for 24 hours, followed by rTNFα treatment. AML were then cocultured with DNTs for 24 hours (for KG-1a) or 2 hours (for OCI-AML2). Percentage increase in specific killing after rTNFα pretreatment was determined. The data shown are representative of 2 independent experiments. (B-C) WT OCI-AML2 and OCI-AML3 cells were untreated or transduced with shRNAs against JAK1 (shJAK1) or GFP (shGFP). RNA expression of JAK1 was normalized to HPRT housekeeping gene (B). Transduced AML cells were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs for 2 hours. Specific killing (left) and percentage increase in specific killing (right) after rTNFα pretreatment are shown (C). (D) AML cell lines (top), primary AML samples with patient IDs, and healthy PBMCs (bottom) were untreated (blue) or treated (orange) with rTNFα. Representative histograms of ICAM-1 expression with MFI values including fluorescence-minus-one (FMO) control (red) are shown. (E) CD33+CD45lowCD3−CD34+ (CD34+) and CD33+CD45lowCD3−CD34− (CD34−) primary AML blasts from 2 patients were treated with rTNFα. The ICAM-1 MFI fold change is shown. (F) ICAM-1 expression with MFI values of AML cell lines cocultured with or without DNTs for 24 hours in the presence of anti-TNFα or isotype control. The data shown are representative of 2 independent experiments. (G) MV4-11 and KG-1a were cocultured with or without DNTs in separate compartments of the transwell from Figure 4B. Representative histograms of ICAM-1 expression on KG-1a with MFI values after 2 days is shown. (H) KG-1a cells were alone or were incubated with MV4-11 or OCI-AML3 and DNTs in the presence of anti-TNFα blocking antibody or isotype control for 24 hours. ICAM-1 expression on live KG-1a cells was determined. The data are representative of 2 independent experiments. (I) KG-1a and OCI-AML2 were treated with JAK1 inhibitor or DMSO for 24 hours, followed by stimulation with or without rTNFα and stained for ICAM-1. The MFI fold change of ICAM-1 expression from rTNFα-treated AML cells relative to untreated AML cells is shown and compared between vehicle and JAK1 inhibitor conditions. The data displayed are 2 pooled independent experiments. (J) JAK1 knockdown (shJAK1) or control (shGFP) AML cells were untreated or treated with rTNFα (100 ηg/mL), then stained with anti–ICAM-1 antibody. The MFI fold change of ICAM-1 expression from rTNFα-treated AML cells relative to untreated AML cells is shown and compared between control and JAK1 knockdown conditions. The data shown are representative of 2 independent experiments. Student t test and 1-way/2-way ANOVA were used. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; shRNA, short hairpin RNA.

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