For ADCC activity, rituximab- or obinutuzumab-opsonized or non-opsonized 51Cr-labeled Raji were used as targets. For redirected killing assay, NK cells were incubated with FcR+ P815 targets in the presence of anti-CD16, anti-NKp46, anti- NKp30 or anti-NKG2D mAbs.
Maximal and spontaneous release was obtained by incubating 51Cr-labeled individual targets, as used for each stimulation condition, with SDS 10% or medium alone, respectively. The percentage of specific lysis was calculated according to the formula: percentage of specific release = (experimental-spontaneouns release)/(total-spontaneous release) × 100. Lytic units for 1 × 106 effector cells were calculated as follows: 1 × 106 /(no. of target cells × X), where X is the E:T ratio resulting in 10% specific lysis.
For IFNγ release, anti-CD20-experienced NK cells were mixed (2:1) with the indicated targets in the presence or absence of recombinant human IL-12 (10 ng/mL) (Peprotech, code 200–12) or IL-2 (100 U/mL) (R&D Systems, code 202-IL), for 18 h. Supernatants were collected and analyzed by commercial ELISA kit (Thermo Scientific, code EHIFNG2), according to the manufacturer's instructions.
Maximal and spontaneous release was obtained by incubating 51Cr-labeled individual targets, as used for each stimulation condition, with SDS 10% or medium alone, respectively. The percentage of specific lysis was calculated according to the formula: percentage of specific release = (experimental-spontaneouns release)/(total-spontaneous release) × 100. Lytic units for 1 × 106 effector cells were calculated as follows: 1 × 106 /(no. of target cells × X), where X is the E:T ratio resulting in 10% specific lysis.
For IFNγ release, anti-CD20-experienced NK cells were mixed (2:1) with the indicated targets in the presence or absence of recombinant human IL-12 (10 ng/mL) (Peprotech, code 200–12) or IL-2 (100 U/mL) (R&D Systems, code 202-IL), for 18 h. Supernatants were collected and analyzed by commercial ELISA kit (Thermo Scientific, code EHIFNG2), according to the manufacturer's instructions.