Figure 5.

Lymphoma- and myeloma cell-restricted activation of CD95 by antibody-scBaff fusion proteins. (a) HT1080 were cultivated in 96-well plates and were challenged in the presence of 2.5 µg/ml CHX with 4 × 10⁴ L363, BJAB or Jurkat cells along with αCD95_N297A-scBaff and αCD95_Fab2-scBaff. Next day, remaining viable plastic adhered cells were quantified by crystal violet staining. (b) HT1080 cell were again seeded in 96-well plates and were supplemented the next day as indicated with 4 × 10⁴ L363, BJAB or Jurkat cells. Co-cultures were pretreated for 30 min with or without 5 µg/ml Baff-TNC and were then stimulated with 20 ng/ml of the αCD95-antibody scBaff fusion proteins, respectively. On the next day, CD95 activation was evaluated by determination of cellular viability. (c) Cocultures of HT1080 with 4 × 10⁴ MM.1S, L363, BJAB or Jurkat cells were cultivated in 96-well plates and were challenged in the presence of 2.5 µg/ml CHX and 20 µM zVAD along with αCD95_N297A-scBaff and αCD95_Fab2-scBaff. On the next day, NFκB signaling was assessed by means of IL8 ELISA. (d) Co-cultures were set up as in “C” and were pretreated for 30 min with or without 5 µg/ml Baff-TNC. Cells were then stimulated with 20 ng/ml of the αCD95-antibody scBaff fusion proteins and on the next day CD95-mediated IL8 production was again assayed by ELISA.