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L. Thurner et al.
and viral pathogens, revealed no significant binding of the pooled DLBCL-BCR.
Confirmation of Ars2, sumoylated JmjD4, and ubiquitinated FamH3 as targets of diffuse large B-cell lymphoma B-cell receptors and determination of the B-cell receptor-binding epitope
ELISA with recombinant Ars2 (UnigeneID: Hs.111801) expressed with a C-terminal FLAG-tag in HEK293 con- firmed Ars2 as the BCR target antigen from three of five (60%) ABC-derived cell lines (OCI-Ly3, OCI-Ly10, and
A
U2932; but not HBL1 and TMD8) and none of four GCB- DLBCL cell lines (Figure 1A). Recombinant BCR from two of 11 DLBCL cases (with unknown cell of origin), but none of nine mantle cell lymphomas and none of 11 pri- mary central nervous system lymphomas were reactive with Ars2 (Figure 1B). Of the two Ars2-reactive DLBCL, one was a non-GCB type and one was unclassified according to immunohistochemistry using the Hans clas- sifier.21 ELISA with fragments of different lengths of Ars2 as the coat identified a region spanning amino acids 350 to 416 as the BCR-binding epitope (Figure 1C), and all
B
Figure 1. Reactivity of diffuse large B- cell lymphoma-derived B-cell receptors
C
(A) Enzyme-linked immunosorbent assay (ELISA) for reac- tivity against Ars2 or sumoylated HSP90 as control of diffuse large B-cell lym- phoma (DLBCL) cell line-derived natural (Papain-digested) B-cell receptors (BCR) in the Fab format and directly for cell membrane-bound BCR. The columns represent adsorbance at an optical den- sity (OD) of 490 nm (mean and standard deviation). (B) ELISA for Ars2 reactivity of recombinant BCR derived from primary DLBCL cryospecimens and controls. The columns represent adsorbance at OD 490 nm (mean and standard deviation). (C) Determination of the affinity region of BCR against Ars2: The affinity region within Ars2 was amino acids (AA) 301- 416 with the highest observed affinity shown for AA 350-416. The columns rep- resent adsorbance at OD 490 nm, (mean and standard deviation). ABC: activated B-cell type; GC: germinal cen- ter B-cell type. (Figure continued on the
against Ars2.
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haematologica | 2021; 106(8)