Fig. 1: The induction of CLL cell proliferation by engineered HS5 cell lines.

A Primary CLL cells (CLL_091) were incubated for 1 h with conditioned media from HS5-WT, HS5-IL21, HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells. Activation of IL21 signaling was detected by phosphorylation of STAT1 and STAT3. GAPDH was used as a loading control. B Primary CLL cells (CLL_065) co-cultured (2 h) on HS5-WT, HS5-IL4, HS5-CD40L, HS5-CD40L^LOW-IL4 or HS5-CD40L-IL4 cells. Downstream effectors of the IL4 receptor (STAT6) and CD40L (IKKs) were detected by immunoblotting. C Bright-field image of HS5-WT and HS5-CD40L-IL4-IL21 cells (upper part) and CLL cells co-cultured on indicated HS5 cells (bottom part) for 3 days. Magnification 200×. D Primary CLL cells (n = 16, viably frozen) were labeled with CFSE after thawing and co-cultured (7 days) on engineered HS5 cell lines (Di). Dii Representative examples of CFSE staining from HS5-CD40L-IL4-IL21 co-cultures. For sample characteristics see Supplementary Table S1 (CLL No. 004, 005, 014, 018, 024, 033, 036, 037, 050, 051, 057, 072, 074, 083, 084, 091). E Primary CLL cells (n = 4) were labeled with CFSE and cultured in the presence of indicated cytokines (7 days). F Viability (indicated as the percentage of 7-AAD negative cells) of CLL samples presented in panel D. G Viability of CLL cells (n = 15) co-cultured on HS5-WT or on HS5-CD40L-IL4-IL21 with or without 70 nM β-mercaptoethanol (β-ME) for 2, 5 and 7 days. H Scheme of inserts used for microwell co-culture of CLL with HS5 cell lines. I Percentage of Ki67 positive CLL cells after 14 days of co-culture with HS5-CD40L-IL4 cells in microwells (MiW) or standard 12-well plate. J Viability of CLL cells (n = 4) long-term co-cultured on mitotically inactivated HS5-CD40L-IL4 (dashed line) or HS5-CD40L-IL4-IL21 cells (solid line). CLL cells were collected and plated on fresh layer of supportive cells every 7 days.