Page 202 - 2021_06-Haematologica-web

L. Thurner et al.
A Figure 4. Targeting Ars2-reactive diffuse large B-cell lymphoma with Ars2-containing immunotoxins. (A) Growth inhibition by B-cell receptor (BCR)-antigen/immunotoxins. Growth of U2932 cells was inhibit- ed by addition of Ars2/ETA’, an immunotoxin of the epitope of the cognate antigen Ars2 fused to a truncated form of Pseudomonas aeruginosa exotoxin A. This growth inhibition could be prevented by co- incubation with neutralizing Ars2- reactive Fab but not by LRPAP1- reactive Fab. Columns (formazan formation detected at an optical density of 450 nm) represent cell proliferation (mean and standard deviation). (B) BCR-specific lysis of DLBCL cell lines by Ars2/ETA’ immunotoxin. Above: cytotoxic effect after 24 h, 48 h and 72 h of incubation with 5 mg/mL of recom- binant Ars2/ETA’ or LRPAP1/ETA’ immunotoxins. Cell viability of HBL- 1 (left) and OCI-LY3 (right) cell lines determined by trypan blue staining. Below: Dose-dependent cytotoxic effect of Ars2/ETA’ determined in a lactate dehydrogenase (LDH)
B release assay. Curves indicate per- cent specific lysis of the HBL1 line (left) or OCI Ly3 line (right) with and without Ars2-reactive BCR, after incubation with doses from 0 μg/mL to 10 mg/mL Ars2/ETA’, LRPAP1/ETA’ or phosphate- buffered saline. (C) Induction of apoptosis by addition of Ars2/ETA’ immunotoxins. Flow cytometric characterization of apoptotic U2932 or HBL1 cells 24 h after addition of Ars2/ETA’ or MAZ/ETA’ by annexin-V/propidium iodide staining. U2932 cells have Ars2- reactive BCR resulting in a strong increase of early and late apoptotic
C
cells after addition of Ars2/ETA’.
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haematologica | 2021; 106(8)