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Ars2 in the pathogenesis of DLBCL
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Figure 1. Continued from previous page. (D) Binding of Ars2 and internalization into U2932 cells. At time 0 (left) Ars2 (a fragment of 253 to 416 amino acids) bound to the cell surface of U2932 cells (above) and was not detected intracellularly (below). After 60 min incubation at 37°C (right) and washing, Ars2 was no longer detected on the cell surface (above), but was found intracellularly (below). Non-specific binding was determined by incubation with antibodies alone. (E) ELISA for reactivity against Ars2 of the BCR of OCI-Ly10 wt and K98A. Recombinant and papain-digested (natural) Fab of OCI-Ly10 bound to Ars2. K98A mutagenesis of recom- binant OCI-Ly10 BCR resulted in the loss of reactivity of the recombinant BCR against Ars2. K98A had previously been described for OCI-Ly10 as being responsible for loss of BCR autoreactivity against apoptotic debris. The columns represent adsorbance at OD 490 nm (mean and standard deviation).
Ars2-specific BCR derived from cell lines and cryospeci- mens bound to this epitope. ELISA with recombinant sumoylated JmjD4 or ubiquitinylated FamH83 expressed with a C-terminal FLAG-tag in HEK293 confirmed each as target antigens of one DLBCL-derived recombinant Fab and ubiquitinated FamH83 in addition as a target of a recombinant mantle-cell lymphoma–derived Fab (Online Supplementary Figures S1 and S2). Fab did not bind to non- modified JmjD4 and FamH83. Binding of Ars2 to mem- brane BCR was demonstrated by flow cytometry of DLBCL cell lines with Ars2-reactive BCR, but not for the TMD8 line without Ars2-reactive BCR. Internalization of C-terminally FLAG-tagged Ars2 into U2932 cells was observed after 60 min (Figure 1D). For OCI-Ly10, muta- genesis of K98A of the BCR heavy chain gene had been reported to result in loss of autoreactivity.8 OCI-Ly10 K98A resulted in loss of affinity against Ars2 (Figure 1E).
Characterization of Ars2 in diffuse large B-cell lymphoma with Ars2-specific B-cell receptors
No obvious differences in molecular weight of Ars2 from DLBCL cases with Ars2-reactive and Ars2-non-reac- tive BCR and controls were observed in western blots; similarly, Sanger sequencing revealed identical DNA sequences, excluding mutations in the coding sequence as an explanation for the immunogenicity of Ars2. However, isoelectric focusing of cell lines (Figure 2A) and DLBCL cases (Figure 2B) with an Ars2-reactive BCR revealed a less negatively charged Ars2 isoform. Dephosphorylation with alkaline phosphatase treatment led to a stronger reduction of the negative charges of Ars2 from DBLCL cases and cell lines without Ars2-specific BCR than in cases with Ars2-reactive BCR and resulted in the disappearance of the differences in electric charge
between both isoforms of Ars2 (Figure 2C), demonstrat- ing that the different isoelectric focusing pattern of Ars2 was due to hypophosphorylation in cases with Ars2-spe- cific BCR. The hypophosphorylated Ars2 isoform was detected in all of the three DLBCL cell lines with Ars2- reactive BCR (i.e., OCI-Ly3, OCI-Ly10, and U2932), but in none of six DLBCL lines without Ars2-reactive BCR. This association in the nine analyzed DLBCL cell lines between BCR reactivity against Ars2 and the presence of the hypophosphorylated isoform of Ars2 was statistically significant (Fisher exact t-test: two-tailed P=0.0119). Regarding the 11 DLBCL cases with recombinantly expressed Fab (Online Supplementary Table S1) derived from cryospecimens, only the two cases with Ars2-reac- tive Fab (#3 and #11) showed hypophosphorylation of Ars2. In this cohort of 11 cases of cryospecimens and recombinantly expressed Fab, the association between Ars2 reactivity and presence of hypophosphorylated Ars2 isoform was also statistically significant (Fisher exact t- test: two-tailed P=0.0182). Looking at the association between BCR reactivity of DLBCL cells and the presence of hypophosphorylated Ars2 isoform overall, considering data from both cell lines and cases with cryospecimen- derived recombinant Fab, the association was statistically highly significant (Fisher exact t-test: two-tailed P<0.0001).
Hypophosphorylated Ars2 was detected in the biopsies of eight of 31 (26%) ABC-type DLBCL cases character- ized by GEP, but in only one of 20 (5%) GCB-type DLBCL and in the peripheral blood from one in 100 healthy controls.
The hypophosphorylated sites were identified as serine 328 and serine 341 by site-directed point mutagenesis of various predicted sites in Ars2 transfected with a C-termi-
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