hAPRIL0.1A prevents APRIL binding to lymphoma cell lines and its survival effect on peripheral blood–derived CLL cells. (A) Five different lymphoma cell lines are shown to bind APRIL (black line) and this effect is blocked by hAPRIL0.1A (black dashed line). (B) The highest TACI/BCMA expressing cell lines (RAJI and L363, supplemental Figure 2) were screened for receptor detection after 24 hours incubation in the absence or presence of FLAG-hAPRIL. Loss of receptor detection after FLAG-hAPRIL stimulation is fully prevented when hAPRIL.1A is added to the overnight culture. (C) TACI and BCMA in cytosine guanine dinucleotide treated CLL (CLL 2, supplemental Figure 3) are down-regulated by the binding of FLAG-hAPRIL. hAPRIL.01A (hA.01A) blocks FLAG-hAPRIL binding and therefore blocks receptor down regulation of TACI and BCMA. BR-3 is taken along as a nonAPRIL binding control. (D) cytosine guanine dinucleotide treated CLL cells expressing both TACI and BCMA (CLL 2, supplemental Figure 3) were cultured in the absence or presence of FLAG-hAPRIL and tested for cell survival at different time-points. The effect of hAPRIL.01A was analyzed on control (MOCK) and FLAG-hAPRIL treated cells (APRIL). Each sample was analyzed in triplicate; ***P < .001 1-way ANOVA; error bars = SD. (E). Apoptosis of CLL cells measured after 3 and 8 days in the absence or presence of APRIL and in the presence of APRIL plus hAPRIL01.A. Apoptosis is measured using annexinV staining and counterstaining for dead cells (late apoptotic) with 7-AAD. (F). Representative pictures (magnified 5×) of the cells treated as indicated in panel D. Experiments (A-C) are accompanied by histograms that represent the relative mean fluorescence intensity (MFI) determined by the ratio between the background and the specific MFI. Basal value (MFI = 1) is pointed out by the gray dotted line.