American Society of Hematology

Figure 2.

$Figure 2. ADCC with donor-derived MNCs, obtained from patients after transplantation. (A) ADCC against MHH4 target cells mediated by CD19-4G7chim. Effector cells from the majority of patients produced enhanced specific lysis of MHH4 target cells, whereas a minor fraction did not. For greater clarity, the patients (n = 15) have been separated into 2 groups: patients with effector cells producing more than 10% (n = 10, group II) or less than 10% (n = 5, group I) specific lysis with CD19-4G7chim and IL-2 stimulation (E/T ratio = 20:1). The mechanism underlying this differential response is still unknown. In group II, both unstimulated and IL-2–stimulated effector cells elicited significantly enhanced lysis after addition of CD19-4G7chim (P < .01 for E/T ratio = 20:1 in both cases, paired Wilcoxon rank sum test). Each patient is represented by connected data points (without or with antibody). Median values are shown as black bars. Effector cells from patients in group I produced no substantial ADCC lysis (< 10%). Specific lysis was measured at varying E/T ratios. Only values obtained at E/T = 20:1 are shown. (B) ADCC against MHH4: comparison of CD19-4G7chim and anti-CD20–mediated effects. Specific lysis by effector cells from patients in group II (A) at various E/T ratios. Data points are mean values of specific lysis averaged over experiments with MNCs from 5 (Mabthera) or all 10 patients (CD19-4G7chim). Antibodies were used in equivalent concentrations. (C) ADCC against cryopreserved primary B-lineage ALL blasts with CD19-4G7chim. ADCC with MNCs from 7 patients against allogeneic (CD19+CD20–) cALL blasts (obtained from 2 other patients) and with MNCs from 1 patient against autologous cALL blasts. To increase reliability of the results, 2 to 4 independent experiments were performed with effector cells from each patient. Mean values of 22 experiments are shown. MNCs of each patient produced significantly enhanced lysis after addition of CD19-4G7chim (P = .026 unstimulated, P < .001 stimulated; paired Wilcoxon rank sum test). (D) Specificity controls and blocking of HLA class I on primary B-lineage ALL blasts. Data points are mean values obtained with IL-2–stimulated MNCs at E/T = 20:1, and bars 2 and 3 represent the same data given in panel C for E/T = 20:1. n = number of experiments used to calculate mean values. Blocking of HLA class I on leukemic blasts with antibody W6/32 (α HLA-I) increased lysis to values reached with CD19-4G7chim, suggesting that CD19-4G7chim mediated ADCC despite the possible presence of HLA class I–mediated inhibitory signals. Differences between MNC and (MNC + anti-CD19) or (MNC + anti–HLA I) were highly significant with P < .001 and P < .005, respectively (paired Wilcoxon rank sum test). As expected, neither the parental murine 4G7 antibody nor the chimeric anti-CD20 IgG1, used as specificity control, induced enhanced target cell lysis.$

ADCC with donor-derived MNCs, obtained from patients after transplantation. (A) ADCC against MHH4 target cells mediated by CD19-4G7chim. Effector cells from the majority of patients produced enhanced specific lysis of MHH4 target cells, whereas a minor fraction did not. For greater clarity, the patients (n = 15) have been separated into 2 groups: patients with effector cells producing more than 10% (n = 10, group II) or less than 10% (n = 5, group I) specific lysis with CD19-4G7chim and IL-2 stimulation (E/T ratio = 20:1). The mechanism underlying this differential response is still unknown. In group II, both unstimulated and IL-2–stimulated effector cells elicited significantly enhanced lysis after addition of CD19-4G7chim (P < .01 for E/T ratio = 20:1 in both cases, paired Wilcoxon rank sum test). Each patient is represented by connected data points (without or with antibody). Median values are shown as black bars. Effector cells from patients in group I produced no substantial ADCC lysis (< 10%). Specific lysis was measured at varying E/T ratios. Only values obtained at E/T = 20:1 are shown. (B) ADCC against MHH4: comparison of CD19-4G7chim and anti-CD20–mediated effects. Specific lysis by effector cells from patients in group II (A) at various E/T ratios. Data points are mean values of specific lysis averaged over experiments with MNCs from 5 (Mabthera) or all 10 patients (CD19-4G7chim). Antibodies were used in equivalent concentrations. (C) ADCC against cryopreserved primary B-lineage ALL blasts with CD19-4G7chim. ADCC with MNCs from 7 patients against allogeneic (CD19⁺CD20^–) cALL blasts (obtained from 2 other patients) and with MNCs from 1 patient against autologous cALL blasts. To increase reliability of the results, 2 to 4 independent experiments were performed with effector cells from each patient. Mean values of 22 experiments are shown. MNCs of each patient produced significantly enhanced lysis after addition of CD19-4G7chim (P = .026 unstimulated, P < .001 stimulated; paired Wilcoxon rank sum test). (D) Specificity controls and blocking of HLA class I on primary B-lineage ALL blasts. Data points are mean values obtained with IL-2–stimulated MNCs at E/T = 20:1, and bars 2 and 3 represent the same data given in panel C for E/T = 20:1. n = number of experiments used to calculate mean values. Blocking of HLA class I on leukemic blasts with antibody W6/32 (α HLA-I) increased lysis to values reached with CD19-4G7chim, suggesting that CD19-4G7chim mediated ADCC despite the possible presence of HLA class I–mediated inhibitory signals. Differences between MNC and (MNC + anti-CD19) or (MNC + anti–HLA I) were highly significant with P < .001 and P < .005, respectively (paired Wilcoxon rank sum test). As expected, neither the parental murine 4G7 antibody nor the chimeric anti-CD20 IgG1, used as specificity control, induced enhanced target cell lysis.

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