Figure 1.
Flow cytometry–based HTS assay identifies the involvement of CD120b/TNFR pathway in DNT-mediated cytotoxicity. (A) Schematic of the flow cytometry–based HTS assay using DNT-resistant (KG-1a) and -susceptible (OCI-AML3) AML cell lines. (B) DNTs alone or cocultured with OCI-AML3 or KG-1a in 96-well plates were stained with 385 fluorophore-conjugated antibodies. Molecule expression on DNTs (CD3+CD33–CD34–) cocultured with OCI-AML3 (CD3–CD33+CD34–) or KG-1a (CD3–CD33–CD34+) relative to DNT alone was determined. From left to right, the graph shows molecules that are upregulated or downregulated on DNTs during the interaction with OCI-AML3, relative to DNT-KG-1a interactions. The experiment was performed with 3 biological replicates, and the data were pooled together. (C) DNTs stained for CD120b after a 2-hour coculture with DNT-resistant (KG-1a) or -susceptible (OCI-AML2, OCI-AML3, and MV4-11) AML cell lines. A representative histogram (left) and corresponding median fluorescence intensity (MFI) values (right) of CD120b expression are shown. Experiments were done in triplicates. The data shown are representative of 3 independent experiments. (D) DNTs were cocultured with DNT-susceptible AML cell lines, OCI-AML2 (top) or MV4-11 (bottom), for 24 hours in the presence of anti-CD62ε or anti-CD120b neutralizing antibody or corresponding isotype controls. Experiments were done in triplicates. The data shown are representative of 2 independent experiments. (E) Linear regression analysis performed between the MFI fold change in CD120b expression on DNTs cocultured with AML cells relative to DNT alone and the percentage specific killing of AML cell lines and primary AML samples by DNTs. AML cells and DNTs were coincubated for 2 hours. Each symbol represents an AML cell line or primary AML sample. Numbers represent the ID of patients with AML. Experiments were done in triplicates, and the data shown are representative of 2 independent experiments. Student t test, 1-way analysis of variance (ANOVA), and linear regression analysis were used. ∗∗∗P < .001. ns, nonsignificant.

Flow cytometry–based HTS assay identifies the involvement of CD120b/TNFR pathway in DNT-mediated cytotoxicity. (A) Schematic of the flow cytometry–based HTS assay using DNT-resistant (KG-1a) and -susceptible (OCI-AML3) AML cell lines. (B) DNTs alone or cocultured with OCI-AML3 or KG-1a in 96-well plates were stained with 385 fluorophore-conjugated antibodies. Molecule expression on DNTs (CD3+CD33–CD34–) cocultured with OCI-AML3 (CD3–CD33+CD34–) or KG-1a (CD3–CD33–CD34+) relative to DNT alone was determined. From left to right, the graph shows molecules that are upregulated or downregulated on DNTs during the interaction with OCI-AML3, relative to DNT-KG-1a interactions. The experiment was performed with 3 biological replicates, and the data were pooled together. (C) DNTs stained for CD120b after a 2-hour coculture with DNT-resistant (KG-1a) or -susceptible (OCI-AML2, OCI-AML3, and MV4-11) AML cell lines. A representative histogram (left) and corresponding median fluorescence intensity (MFI) values (right) of CD120b expression are shown. Experiments were done in triplicates. The data shown are representative of 3 independent experiments. (D) DNTs were cocultured with DNT-susceptible AML cell lines, OCI-AML2 (top) or MV4-11 (bottom), for 24 hours in the presence of anti-CD62ε or anti-CD120b neutralizing antibody or corresponding isotype controls. Experiments were done in triplicates. The data shown are representative of 2 independent experiments. (E) Linear regression analysis performed between the MFI fold change in CD120b expression on DNTs cocultured with AML cells relative to DNT alone and the percentage specific killing of AML cell lines and primary AML samples by DNTs. AML cells and DNTs were coincubated for 2 hours. Each symbol represents an AML cell line or primary AML sample. Numbers represent the ID of patients with AML. Experiments were done in triplicates, and the data shown are representative of 2 independent experiments. Student t test, 1-way analysis of variance (ANOVA), and linear regression analysis were used. ∗∗∗P < .001. ns, nonsignificant.

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