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Ars2 in the pathogenesis of DLBCL
stimuli of different subgroups of DLBCL.3,4 In particular, DLBCL of the ABC-type or the recently specified MCD- type or Cluster 5 harbor recurrent mutations in MYD88 and CD79B genes with dependency on constitutive BCR signal- ing.5-8 For primary central nervous system lymphoma, which represents a specific extranodal subtype of DLCBL with molecular similarities to MCD type or C5 with fre- quent mutations in MYD88 and CD79,9,10 SAMD14/neura- bin-I were recently identified as antigens of BCR, and were hyper-N-glycosylated specifically in patients with SAMD14/neurabin-I-reactive BCR.11 In systemic DLBCL, a cis and trans stimulation of the BCR by a so far uncharacter- ized autoantigen was reported for the HBL1 line. Moreover, an anti-idiotype reactivity of complementarity determining region 3 of the BCR of the TMD8 line against an epitope within its own FR2 (V37R38) was described, and for U2932 and OCI-LY10 lines BCR reactivity against apoptotic cell debris was reported.8 This prompted us to screen for and characterize possible target antigens of BCR of systemic DLBCL using expression cloning of primary cryopreserved specimens and DLBCL lines and subsequent protein array screening.12-14
Methods
The study was approved by the local ethics committee (Ärztekammer des Saarlandes 12/13). For expression cloning of DLBCL-BCR, patients’ snap-frozen specimens were obtained from the Dr. Senckenberg Institute of Pathology (Frankfurt am Main, Germany). Sera of a second cohort of patients with DLBCL were obtained from the DSHNHL RICOVER-60 trial.
DLBCL cryospecimens, of a third cohort of patients, of which the cell-of-origin had been determined by GEP, were obtained from the Institutes of Pathology of Würzburg and Kiel Universities.
B-cell receptor screening for autoantigens
BCR from nine DLBCL cell lines were prepared by digestion with papain. Moreover, expression cloning of recombinant BCR derived from primary DLBCL cryospecimens was performed, as described in the Online Supplementary Material. These DLBCL line- derived BCR and the pooled recombinantly expressed BCR (each at a concentration of 10 mg/mL) were screened on protein macroarrays containing clones of UniPEx 1 and 2 cDNA expres- sion libraries (Bioscience, Dublin, Ireland), as previously described.13,14 To search for further antigens, all recombinant DLBCL-derived antigen-binding fragments (Fab) without reactivi- ty against arsenite resistance protein 2 (Ars2) were screened against variously post-translationally modified UniPEx 1 and 2 protein macroarrays, including sumoylation, ubiquitination, citrul- lination, and acetylation. Protein macroarrays were sumoylated as described elsewhere15 and ubiquitination was performed with synchronized HeLa cell extracts.16 The screening for antigens of infectious origin is described in the Online Supplementary Material.
Expression of target antigens and immunotoxins
The expression clone of Ars2 and subsequently the epitope- containing region consisting of amino acids 342-375 of Ars2 were recombinantly expressed with a C-terminal FLAG tag by a pSFI vector in HEK293 cells. Additionally, C-terminally FLAG-tagged full-length Ars2 was transfected by electroporation into U2932 and TMD8 via a pRTS vector.17 C-terminally FLAG-tagged FamH83 and JmjD4 were recombinantly expressed in HEK293 cells. Site-directed mutagenesis of Ars2 and secondary modifica-
tion of antigens is described in the Online Supplementary Material. Immunotoxins with monomethyl auristatin E (MMAE) are effective in vivo and established in the clinics, but the synthesis of toxin conjugates with MMAE requires enzyme-cleavable dipep- tide linkers and is therefore challenging for academic laborato- ries.18,19 Hence, a truncated form of Pseudomonas aeuroginosa exo- toxin A (ETA’) was used, as the ETA’ conjugate can be recombi- nantly expressed directly. Recombinantly expressed immunotox- ins, consisting of Ars2 amino acids 342-375 conjugated to ETA’ were either obtained from the Fraunhofer Institute of Experimental Medicine and Immunotherapy (Aachen, Germany) or recombinantly expressed in our laboratory in E. coli BL21 and
purified by the His-Tag, as described by Nachreiner et al.20
Enzyme-linked immunosorbent assay (ELISA) for B-cell receptor and serum reactivity against target antigens and competition ELISA with apoptotic debris
Ars2, ubiquitinated FamH83, and sumoylated JmjD4 were con- firmed as BCR antigens by ELISA, as previously described.13 ELISA and competition assays with apoptotic debris are described in detail in the Online Supplementary Material.
Western blot and isoelectric focusing
Lysates of DLBCL lines or of whole blood from patients were loaded and separated by 10% sodium dodecylsulfate polyacryl- amide gel electrophoresis and transferred to a polyvinylidene flu- oride membrane using a transblot semidry transfer cell (Bio Rad). After blocking overnight at 4°C in phosphate-buffered saline/10% nonfat dry milk, a recombinant Ars2-reactive His-tagged Fab was incubated at a concentration of 2 mg/mL for 1 h at room tempera- ture, followed by incubation for 1 h at room temperature with murine anti-his antibody at a ratio of 1:2,000 (Qiagen), with horse- radish peroxidase-labeled anti-mouse IgG antibody (Bio Rad). Chemiluminescence reagent (New England BioLabs) was used for immunoblot detection. Isoelectric focusing was performed as pre- viously described. Proliferation, BCR pathway activation assays, cytotoxicity and apoptosis assays are described in the Online Supplementary Material.
Results
Recombinant BCR in the form of Fab were successfully synthesized from 11 DLBCL cases. Moreover, Fab of “nat- ural” BCR were obtained by papain digestion from nine DLBCL cell lines. From three of these cell lines, recombi- nant Fab were generated (Online Supplementary Table S1).
Screening of protein macroarrays and a library of infective agents
The screening of DLBCL Fab identified an expression clone of Ars2 transcript variant 2 (RZPDp828K0526 from Unipex 2, UnigeneID: Hs.111801) spanning from amino acids 253 to 416 as the candidate antigenic target. The screening of the Fab of DLBCL cases and cell lines on post- translationally modified protein macroarrays revealed sumoylated JmJD4 (RZPDp9027E0216D from Unipex 1; UnigeneID: Hs.555974) and ubiquitinylated FamH83 (RZPDp828G0328 from Unipex 2; UnigeneID: Hs.676336) as candidate antigens. The screening against bacterial lysates of 11 bacterial strains did not reveal any specific reactivity. Screening of an Infectious Disease Epitope Microarray (PEPperCHIP® Heidelberg, Germany) consisting of 3,760 database-derived B-cell epitopes associated with 196 pathogens, including various bacterial, fungal, parasitic,
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