Nok 2

Normal human keratinocytes were obtained from foreskin and cultured as described previously (16 (link)). Briefly, normal human keratinocytes were amplified on mitomycin C (Sigma-Aldrich, St Louis, MO, USA)-treated 3T3 cells and cultivated in Dulbecco’s modified Eagle’s medium and Ham’s F12 medium. Subconfluent secondary cultures for experiments were plated in a defined serum-free medium (KGM, Lonza Walkersville Inc., Walkersville, MD, USA). When cells were confluent, Ca²⁺ concentration was increased to 1.8 mM for 24 h, to induce keratinocyte differentiation, before the addition of any stimulus. Either human recombinant soluble FasL (rFasL) (0.1, 10, or 50 ng/ml, Sigma) or PVIgG and NIgG (1.5 mg/ml) were diluted in keratinocyte basal medium (Lonza) plus 1.8 mM Ca²⁺ and cycloheximide (Sigma) 1 µg/ml, and incubated for a further 72 h. For in vitro inhibitory experiments, keratinocyte cultures were pretreated with either 1 or 15 µg/ml of purified mouse anti-human FasL monoclonal Ab (NOK-2; BD Biosciences Pharmingen, San Diego, CA, USA) for 1 h, which was also added when medium was provided with NIgG, PVIgG, or rFasL.

To inhibit perforin-mediated target cell lysis effector cells were preincubated for 2 h in phenol-free RPMI-1640/10% FBS supplemented with 50 nM concanamycin A (CMA) and 50 nM CMA was also present during the measurements. 1x10⁴ targets per well were re-suspended in 100 µl phenol-free RPMI-1640/10% FBS/50 nM CMA. Effector cells were added in 100 µl of phenol-free RPMI-1640/10% FBS/50 nM CMA after preincubation.
To inhibit granzyme B-mediated target cell lysis, effector cells were preincubated for 2 h in phenol-free RPMI-1640/10% FBS supplemented with 10 µM z-AAD-CMK and 10 µM z-AAD-CMK was also present during the measurement. To this end 1x10⁴ targets per well were submitted in 100 µl of phenol-free RPMI-1640/10% FBS/10 µM z-AAD-CMK. Effector cells were added in 100 µl of phenol-free RPMI-1640/10% FBS/10 µM z-AAD-CMK after preincubation.
To block FasL- and TRAIL-mediated target cell lysis inhibitory anti-human antibodies were used to block the interaction of FasL or TRAIL with their respective apoptosis inducing receptors. To this end 1x10⁴ targets per well and 2x10⁴ effector cells per well were each preincubated for 2 h in 10 µg/ml anti-human CD178 antibodies (NOK-1 and NOK-2, BD Biosciences) or 5 µg/ml anti-human TRAIL antibody (RIK-2, Biolegend). After 2 h of preincubation effector cells were added to target cells.