BCR self-recognition in CLLviaalternative epitopes. (A) Alignment of the insert sequence of the selected epitope mimicking phage YYCYFTEAPYSYWGNLVC with 2 species of Ig identified by protein array screening. Fab007 bound specifically to 2 distinct Igs, BC032452.1 and BC030983.1, on a protein array (ProtoArray human protein microarray; Invitrogen). Sequence homology of the phage insert sequence YYCYFTEAPYSYWGNLVC selected on Fab007 with an epitope within the variable region of Igs BC032452.1 and BC030983.1 is shown. Homologous sequences (single-letter amino acid code) are colored. (B) CLL BCR Fab007 binds to immobilized Igs of the IgG, IgA, and IgM isotype. All Igs and control BSA were coated and incubated with Fab007. Binding of Fab007 was detected by ELISA with an HRP-conjugated anti–His-tag antibody. Data are shown as means from triplicate experiments (± SEM). Sources of Igs: monoclonal Igs of the IgG (κ and lambda) and IgA (κ and lambda) isotype were purified from the sera of multiple myeloma patients by protein-A chromatography for IgG and jacalin chromatography for IgA. Polyclonal IgG and IgM were from Octapharma or USB products, respectively; the Fc fragment of human IgG1 (IgG-Fc) was from R&D Systems (P-Selectin/Fc-gamma, #137-PS). (C) CLL BCR Fab007 displays “self-reactivity.” Fab007 was labeled with biotin and tested for its binding to immobilized unlabeled receptor in an ELISA assay. Bound biotinylated Fab007 (Fab007bio) was detected using alkaline phosphatase–conjugated avidin. Unrelated Fab005 was used as a control (note that this BCR contained the YYC epitope presented herein but not the VRQ epitope described in Dühren-von Minden et al⁸ ). Data are shown as means from triplicate experiments (± SEM). (D) Illustration of the structural basis of potential autostimulatory mechanisms in CLL. Red circles mark potential interactions between BCRs on 2 different CLL cells; blue circles mark potential interactions between BCRs on the same CLL cell. The bottom panels show the structure of Ig heavy chains (gray space-filling model) and Ig light chains (green tube model) with the spatially related VRQ epitope (depicted in violet) and YYC epitope (depicted in dark green). The heavy chain CDR3 region is depicted in orange. Below, the exact positions of both epitopes are shown within the framework regions of Ig heavy (IgHV) and Ig light (IgLV) chains (VRQ epitope in violet; YYC epitope in dark green). For the structural Ig model, a random crystallized Fab fragment from the RCSB database (www.rcsb.org) was chosen (identifier 3GBM).