Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins

Abstract

Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca(2+). Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.

Fig. 1.

Surface IgM-derived μ chains of primary FL B cells carry mannosylated glycans. Viable cells were isolated from diagnostic lymph node biopsies of two FL patients with different numbers of V_H-region N-glycosylation (GLY) sites. Viable normal B cells were purified by negative selection from PBMCs. Cell-surface proteins were isolated from each sample and treated with Endo H or PNGase as indicated. Changes of μ chain mobility after enzyme treatment were analyzed by immunoblotting using an anti-μ antibody. Reproducibility was assessed by repeating the immunoblotting three times with similar results.

Fig. 2.

C-type lectins bind to both primary FL cells and to normal B cells. Lectin binding by live CD19⁺ B cells from two FL cases (A and B) and from one healthy individual (C) was analyzed by FACS. Shaded lines: secondary antibody (anti-human Fcγ) alone; heavy black lines: binding in the presence of Ca²⁺; gray lines: binding in the absence of Ca²⁺. Binding of each individual lectin to FL cells was reproduced twice for each sample. Binding to normal B cells is representative of the same results obtained with B cells from three different donors.

Fig. 3.

Lectin-mediated signaling in FL cells. Cells were labeled with a Ca²⁺-sensitive dye and analyzed by FACS after addition of the indicated reagent at the time point shown by the arrow. B cells were gated using anti-CD19. The y-axis represents percentages of cells over the threshold, set at the 85th percentile of unstimulated CD19⁺ cells. (A) FL29 cells exposed to anti-μ or to lectins, (i) MR, or (ii) DC-SIGN with or without cross-linkage (X). (iii) FL21 exposed to anti-μ or to X-Lectins. (B) Blocking of lectin stimulation of FL29 cells by preincubation of (i) Lectin-MR or (ii) DC-SIGN with mannosylated (Mann) scFv [or control nonmannosylated (Non-Mann) scFv] at a molar ratio of 1:2 (MR) or 1:7 (DC-SIGN). (C) Normal B cells purified from (i) subject 1 exposed to anti-μ or to X-Lectin MR; (ii) subject 2 exposed to anti-μ or to X-DC-SIGN; (iii) PBMCs from subject 3 exposed to anti-μ or to X-Lectin-MR or to X-DC-SIGN. In all cases, anti-μ was F(ab)₂.

Fig. 4.

Lectins block access of anti-μ to sIgM of FL but not of normal B cells. (A) FL cells from two patients, FL29 (i) and FL21 (ii), were exposed to Lectin-MR, to DC-SIGN, or to Control-MR (20 μg/mL) in lectin buffer on ice for 1 h. After washing, cells were treated with F(ab)₂ anti-μ and blockage of binding assessed by FACS. Bound lectins did not block binding of anti-CD19 to FL29 (iii) or to FL21 (iv) cells. (B) PBMCs from a normal subject were exposed to lectins and then to anti-μ as above. (i) B cells showing variable levels of sIgM were unaffected by preexposure to lectins. (ii) Gating on CD27⁺ cells was used to indicate no effect of lectins on sIgM expression by memory B cells. Reproducibility was assessed by repeating the blocking assay three times with similar results. The total number of N-glycosylation sites (GLY) in the IGHV and IGLV genes of each case is indicated.

Fig. 5.

FL-derived mannosylated (Mann) scFv binds to immature dendritic cells via DC-SIGN. Dendritic cells were prepared from blood monocytes and tested for binding of mannosylated scFv-Cκ or nonmannosylated (Non-Mann) scFv-Cκ by FACS using anti-Cκ| for detection. (A) Binding of mannosylated scFv was specific and Ca²⁺ dependent. (B) Dendritic cells with minimal levels of DC-SIGN after knockdown (R1 gate in Fig. S4ii) were tested for binding of mannosylated scFv from two FL patients by FACS. Result shown is representative of three independent experiments. Shaded lines: anti-Cκ| alone; heavy black lines: binding to knocked down (R1) cells; dashed lines: binding to dendritic cells treated with control siRNA.