CAL-101 does not show cytotoxicity toward other normal immune cells but alters cytokine production. (A) CD3⁺ T cells and CD56⁺ NK cells (n = 9; each) from healthy volunteers were incubated with or without CAL-101 (0.1-10μM) for 48 hours. Viability was determined by annexin/PI flow cytometry and was calculated relative to time-matched untreated controls. (B) CD3⁺ T cells (n = 12) from healthy volunteers were incubated with or without CAL-101 (0.1-10μM) for 48 hours. Cells were stimulated with an anti-CD3 T-cell activation plate for 24 hours, and IL-6, IL-10, and TNF-α production was measured by ELISA. For CD40L mRNA assay, CD4⁺ T cells from healthy volunteers (n = 4) were incubated with and without various doses of CAL-101 and 5 μg/mL CD28. Cells were then stimulated with an anti-CD3 T-cell activation plate for 48 hours. Real-time polymerase chain reaction analysis was done to determine quantities of CD40L mRNA. (C) CD56⁺ NK cells (n = 8) from healthy volunteers were incubated with or without alemtuzumab, CAL-101, or the combination for 4 hours. IFN-γ production was determined by ELISA. (D) CD56⁺ NK cells (n = 3) from healthy volunteers were used as effector cells for a CLL-cell ADCC assay. NK cells were left untreated or treated with 10μM CAL-101; whereas CLL effector cells were treated with alemtuzumab. IgG indicates immunoglobulin G; and DMSO, dimethyl sulfoxide. Error bars represent the SD from the mean.