Figure 1.
Effect of ATLG on NK cell function and viability in vitro. (A) Lysis of K562 cells by NK cells. After preincubation of isolated NK cells with 0, 2, or 1000 μg/mL ATLG for 24 hours, NK cell–mediated lysis of K562 cells was quantified using the Europium release assay. E:T ratios varied (20:1 and 10:1). Shown are means of 5 independent assays performed in triplicates, bars indicate standard error of the mean (SEM). For analysis of significance, 1-way analysis of variance (ANOVA) test was performed. (B) Effect of ATLG on NK cell viability. Isolated NK cells were incubated for 24 hours with 0, 2, or 1000 μg/mL ATLG, then stained with annexin V/PI and analyzed by FACS. Columns show means of 4 independent assays, and bars show SEM. (C) Effect of sera obtained from ATLG-treated patients on NK cell viability. Isolated NK cells from 3 healthy donors were incubated for 24 hours with sera from 3 patients after treatment with ATLG, then analyzed by annexin V/PI staining. Sera were obtained on day –12 (before first ATLG dose) and on day −8 (after ATLG application). Patients had not received any chemotherapeutic agents until day –8. ATLG serum levels were adjusted to concentrations ranging from 0.25 to 2 μg/mL in in vitro assays. No significant difference of overall cell death rates (annexin V+/ PI+/annexin V+ and PI+) was seen in the presence of ex vivo reconstituted ATLG and patients’ sera, both adjusted to 2 μg/mL ATLG (1-way ANOVA, Tukey multiple comparisons). All experiments were performed in duplicates. Serum from 1 of 3 patients was tested only once. Shown are means with SEM.

Effect of ATLG on NK cell function and viability in vitro. (A) Lysis of K562 cells by NK cells. After preincubation of isolated NK cells with 0, 2, or 1000 μg/mL ATLG for 24 hours, NK cell–mediated lysis of K562 cells was quantified using the Europium release assay. E:T ratios varied (20:1 and 10:1). Shown are means of 5 independent assays performed in triplicates, bars indicate standard error of the mean (SEM). For analysis of significance, 1-way analysis of variance (ANOVA) test was performed. (B) Effect of ATLG on NK cell viability. Isolated NK cells were incubated for 24 hours with 0, 2, or 1000 μg/mL ATLG, then stained with annexin V/PI and analyzed by FACS. Columns show means of 4 independent assays, and bars show SEM. (C) Effect of sera obtained from ATLG-treated patients on NK cell viability. Isolated NK cells from 3 healthy donors were incubated for 24 hours with sera from 3 patients after treatment with ATLG, then analyzed by annexin V/PI staining. Sera were obtained on day –12 (before first ATLG dose) and on day −8 (after ATLG application). Patients had not received any chemotherapeutic agents until day –8. ATLG serum levels were adjusted to concentrations ranging from 0.25 to 2 μg/mL in in vitro assays. No significant difference of overall cell death rates (annexin V+/ PI+/annexin V+ and PI+) was seen in the presence of ex vivo reconstituted ATLG and patients’ sera, both adjusted to 2 μg/mL ATLG (1-way ANOVA, Tukey multiple comparisons). All experiments were performed in duplicates. Serum from 1 of 3 patients was tested only once. Shown are means with SEM.

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