Figure 6.
ICAM-1–LFA-1 interaction is critical for TNFα to sensitize AML to DNT-mediated cytotoxicity. (A-B) AML cell lines (A) and primary AML samples (B) were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs in the presence of isotype control or anti–ICAM-1 blocking antibody. Percentage specific killing of AML cells (A, left) and percentage increase in specific killing between rTNFα-treated AML and nontreated AML (A, right) were determined. The experiments were performed in triplicates with at least 2 DNT donors. Numbers represent the ID of patients with AML. (C) Cas9 and ICAM-1KO OCI-AML2 cells were untreated or treated with rTNFα, washed with PBS, then cocultured with DNTs for 2 hours. The data shown are the percentage increase in specific killing of rTNFα-treated AML cells by DNTs relative to untreated AML cells. The graph shown is representative of 2 independent experiments. (D) DNTs were stained for LFA-1 subunits (red), CD11a and CD18, and compared with FMO control (blue). (E) Representative histogram of random sgRNA control DNTs (sgRND, orange) and CD18KO DNTs (CD18KO, blue) to show CD18 expression relative to FMO control (red). (F) AML cells were pretreated with or without rTNFα, washed with PBS, then cocultured with CD18KO DNTs (CD18KO) or control DNTs (sgRND) for 24 hours (for KG-1a) and 2 hours (for OCI-AML2). The percentage increase in specific killing due to rTNFα pretreatment was determined. The data shown are representative of 2 independent experiments. (G-H) KG1a (G) and 4 primary AML samples (H) were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs, in the presence of isotype control or anti-CD18 blocking antibody for 24 hours (for KG-1a) and 3 hours (for primary AML). Percentage change in specific killing between rTNFα-treated AML and nontreated AML is shown. The experiments were performed in triplicates with at least 2 DNT donors. Numbers represent the ID of patients with AML. Student t test and 1-way/2-way ANOVA were used. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; sgRNA, single guide RNA; sgRND, random single guide RNA.

ICAM-1–LFA-1 interaction is critical for TNFα to sensitize AML to DNT-mediated cytotoxicity. (A-B) AML cell lines (A) and primary AML samples (B) were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs in the presence of isotype control or anti–ICAM-1 blocking antibody. Percentage specific killing of AML cells (A, left) and percentage increase in specific killing between rTNFα-treated AML and nontreated AML (A, right) were determined. The experiments were performed in triplicates with at least 2 DNT donors. Numbers represent the ID of patients with AML. (C) Cas9 and ICAM-1KO OCI-AML2 cells were untreated or treated with rTNFα, washed with PBS, then cocultured with DNTs for 2 hours. The data shown are the percentage increase in specific killing of rTNFα-treated AML cells by DNTs relative to untreated AML cells. The graph shown is representative of 2 independent experiments. (D) DNTs were stained for LFA-1 subunits (red), CD11a and CD18, and compared with FMO control (blue). (E) Representative histogram of random sgRNA control DNTs (sgRND, orange) and CD18KO DNTs (CD18KO, blue) to show CD18 expression relative to FMO control (red). (F) AML cells were pretreated with or without rTNFα, washed with PBS, then cocultured with CD18KO DNTs (CD18KO) or control DNTs (sgRND) for 24 hours (for KG-1a) and 2 hours (for OCI-AML2). The percentage increase in specific killing due to rTNFα pretreatment was determined. The data shown are representative of 2 independent experiments. (G-H) KG1a (G) and 4 primary AML samples (H) were pretreated with or without rTNFα, washed with PBS, then cocultured with DNTs, in the presence of isotype control or anti-CD18 blocking antibody for 24 hours (for KG-1a) and 3 hours (for primary AML). Percentage change in specific killing between rTNFα-treated AML and nontreated AML is shown. The experiments were performed in triplicates with at least 2 DNT donors. Numbers represent the ID of patients with AML. Student t test and 1-way/2-way ANOVA were used. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; sgRNA, single guide RNA; sgRND, random single guide RNA.

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