Effect of IL-6 on responses of CLL cells to resiquimod. (A) Purified CLL cells (2 × 10⁶ cells/ml) were cultured overnight alone or with IL-6 (100 ng/ml) with or without actemra (15 μg/ml). Some cells were then stimulated with resiquimod and membrane TNF-α, and CD83 expression determined by flow cytometry after 4 hours. An example for patient #9 is shown with similar results obtained for 6 other samples. Numbers in the top right quadrants indicate percentages of mTNF-α⁺CD83⁺ cells. (B-C)TLR7 (B) and TNFA (C) transcript levels were determined in the remaining cells by real-time PCR and normalized to HPRT transcripts. The average and standard deviation (SD) of 4 independent experiments for 4 different patient samples are shown. (D-E) Purified CLL cells (10⁴ cells) were cultured alone or with IL-2 and resiquimod in the presence or absence of IL-6 or stromal supernatant with or without actemra or ruxolitinib. Viable cells were counted after 8 to 12 days (D). [³H]thymidine uptake was measured after 48 hours (E). Averages and standard errors from triplicate wells or 3 separate cell counts are shown for 4 different patient samples. IL-6 tolerized CLL cells to TLR7 agonists and slowed the growth of activated CLL cells. *P < .05; **P < .01; ***P < .001. Ru, ruxolitinib.