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Self-antigen recognition by follicular lymphoma B-cell receptors

Kacey L. Sachen, Michael J. Strohman, Jonathan Singletary, Ash A. Alizadeh, Nicole H. Kattah, Chen Lossos, Elizabeth D. Mellins, Shoshana Levy and Ronald Levy

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Article Figures & Data

Figures

  • Figure 1

    Self-reactivity of recombinant FL Igs. Expressed Igs cloned from FL B cells were tested for self-reactivity by an indirect IFA on HEp-2 cells. (A) Summary of the frequency of self-reactive tumor Igs and the cellular localization of recognized antigens. The number of Igs tested is indicated in the center of the pie chart. (B) Examples of HEp-2 IFA staining patterns. Staining patterns of reactive Igs were confirmed in at least 2 independent experiments. Bars represent 25 μm.

  • Figure 2

    HEp-2 reactivity is not dependent on variable region oligomannose glycans. (A) Number of N-glycosylation motifs in heavy (VH) and light (VL) chain variable regions, not encoded by the germline sequence. The number of sequences analyzed is indicated in the center of the pie chart. (B) Tumor Igs from patients 0998 and 0912 were produced in the absence or the presence of tunicamycin to generate Ig lacking N-linked glycans. Tumor Igs were then treated with Endo H or PNGase F to confirm the presence or absence of oligomannose glycans or N-linked glycans, respectively. Proteins were separated by SDS-PAGE and immunoblotted for human IgG. (C) HEp-2 IFA staining patterns of tumor Igs with and without (indicated by a “T”) N-linked glycans. Results are representative of 2 independent experiments. (D) Intracellular flow cytometric titration curve of HEp-2 cells stained with tumor Ig with and without (indicated by a “T”) N-linked glycans. Results are representative of 2 independent experiments.

  • Figure 3

    Identification of myoferlin as a uniquely recognized self-antigen. (A) Silver stain of a 3%-8% Tris-acetate gel of proteins immunoprecipitated (IP) from HEp-2 cell lysate by the indicated recombinant tumor Igs. (−) indicates lanes containing tumor Ig proteins only; IP, lanes containing the immunoprecipitated proteins; Ly, lysate; and B, lysate IP with protein G beads only. The left arrow indicates the 236-kDa protein immunoprecipitated by the tumor Ig of patient 1152; the right arrow points to the location of the tumor Igs. (B) Immunoblotting for myoferlin in immunoprecipitation samples from HEp-2 cell lysate. (C) Immunoblotting for myoferlin in immunoprecipitation samples from 293T cells transfected with recombinant myoferlin. (D) A total of 98 tumor Igs were tested for binding to recombinant myoferlin by ELISA. Myoferlin-HA was immobilized using anti-HA antibodies on lysates from untransfected (left panel) and transfected 293T cells (right panel). Shown is a representative graph of OD405-490 values for 14 different nonbinding patients' tumor Igs (solid lines) compared with tumor Ig for patient 1152 (dotted line). The ability of tumor Ig 1152 to bind myoferlin was confirmed in at least 2 independent experiments.

  • Figure 4

    Igs of 1152 tumor subclones retain self-reactivity and antigen binding. (A) HEp-2 IFA staining pattern of tumor subclone Igs (2E12, 6C12, 4B11, 1G2, and 1E9) were obtained through rescue fusion of cells from the tumor biopsy of patient 1152; 1152 corresponds to the recombinant tumor Ig from patient 1152. Bars represent 25 μm. (B) Immunoblot for myoferlin in immunoprecipitations from lysate of 293T cells transfected with recombinant myoferlin-HA construct. Ly indicates lysate; B, lysate IP with protein G beads only; and 0516, an unrelated tumor Ig. (C) Tumor subclone Igs were tested for binding to recombinant myoferlin by ELISA. Recombinant Myoferlin-HA protein was immobilized using anti-HA antibodies on lysate from transfected 293T cells. Data shown are representative of at least 2 independent experiments.

  • Figure 5

    Antigen binding by tumor cells of patient 1152 correlates positively with BCR expression. A single-cell suspension of the tumor biopsy was analyzed by flow cytometry to assess antigen binding by individual cells. Recombinant myoferlin with an HA tag or fused to mouse IgG2a FC was used to stain the cells. Antigen binding was detected with goat anti-HA or goat anti–mouse IgG2a. Lysate from untransfected 293T cells served as a negative control (UT). Cells are gated on CD3CD20+ B cells; tumor B cells and nontumor B cells were identified with CD20hiIgM and CD20intIgM+ gates, respectively.

  • Figure 6

    Myoferlin stimulates phosphorylation of S6 ribosomal protein in tumor cells. A single-cell suspension of the tumor biopsy was stimulated with 100 μL of detergent-adsorbed lysate of either untransfected 293T cells (UT), or 293T cells transfected to express recombinant myoferlin containing an HA tag or myoferlin fused to mouse IgG2a Fc. A total of 10 μg/mL of goat anti-IgG and IgM was used as positive control for BCR signaling for tumor and nontumor B cells, respectively. Cells were stimulated for 45 minutes at 37°C. Cells were then fixed with 1.6% paraformaldehyde and permeabilized with methanol. Cells were stained for expression of CD3, CD20, and phosphorylated S6 ribosomal proteins. Tumor and nontumor B cells were identified with CD3CD20hi and CD3CD20int gates, respectively. Values adjacent to histograms indicate median fluorescent intensities.