The Functional Role of Acquired N-Linked Glycosylation Sites On Follicular Lymphoma B Cell Antigen Receptors.

Abstract 2704

Follicular lymphoma (FL) is an indolent B-cell lymphoma characterized by apoptosis resistance due to overexpression of Bcl-2 as a consequence of the t(14;18) translocation, ongoing somatic hypermutation (SHM), and expression of B-cell receptors (BCR) with glycosylation of the antigen binding sites. Translocation and concomitant Bcl-2 overexpression can be found in healthy human blood B cells and is insufficient to drive lymphoma outgrowth in mouse models. Since most FL cells still express a surface B cell receptor (BCR) despite the disruption of one immunoglobulin heavy chain allele by the t(14;18) translocation, expression of an antigen receptor seems to be indispensable for FL development. Around 80% of FLs possess asparagine (N)-linked glycosylation sites (amino acid sequence: N-X-S/T) in their BCR variable regions that are not encoded in germ-line but are acquired through SHM. In contrast to germ-line-encoded glycosylation sites in the constant BCR region, where normal processing of the glycans results in termination on branched sugars like sialic acid, the variable region glycosylation sites carry mannose-terminating sugars. Recently, it has been shown that C-type lectins bind to and stimulate FL cells. Such lectins are normally expressed on cells of the innate immune system, e.g. dendritic cells (DCs), which also reside in close interaction with the transformed B cells in germinal centers. Importantly, previous studies point to an outstanding role of the tumor microenvironment in survival and proliferation of the FL cells.

In this study, we demonstrate that the variable region glycosylation in FL BCRs contribute to stimulation of the cells as well as adhesion to cells of the innate immune system. The BCR from six FL and the appropriate glycosylation-defective controls in which the N-linked glycosylation sequons are removed by replacing the asparagine (N) residues with glutamine (Q) residues were expressed in the tko cellular reconstitution system. In tko cells, the BCR signaling cascade can be rendered functional at will through a tamoxifen-dependent mutant of the signal transducer SLP-65 (Meixlsperger et al., Immunity 2007; Dühren von Minden et al., Nature 2012). Tko cells expressing FL BCRs and their glycosylation-defective controls were tested for binding of a recombinant DC-SIGN/Fc fusion protein by flow cytometry. The mannosylated FL-derived BCR but not glycosylation-mutated receptors bound DC-SIGN. Stepwise mutation of individual glycosylation sites demonstrated variable contribution to the strength of lectin binding. Despite this specific binding to mannosylated FL BCRs, DC-SIGN/Fc failed to induce significant calcium mobilization of transduced tko cells. Crosslinking with anti-IgM, in contrast, led to a readily detectable BCR-mediated signal, thereby demonstrating functionality of the transduced BCR.

To study the role of mannosylated FL receptors in interaction with their environment, we co-cultured cells expressing FL receptors containing or lacking N-linked glycans in the variable regions together with macrophages. Western blot analyses with a pan-phosphotyrosine antibody demonstrated higher global tyrosine phosphorylation in the lysates of cells expressing glycosylated receptors, thereby indicating a specific role for mannosylated V-regions in FL stimulation.

Glycan-mediated interactions fulfill multiple important functions in the mammalian immune system including pathogen recognition and cell adhesion or trafficking. DC-SIGN serves as receptor for the uptake of mannosylated pathogens and contributes to cell-cell interaction by binding to the heavily glycosylated ICAM-2/3 (intracellular adhesion molecules-2/3). In the case of FL, it is therefore conceivable that DC-SIGN expressed on follicular DCs binds to the heavily mannosylated FL BCRs and serves thereby as adhesion molecule to keep the FL B cells within the follicular structure. We tested this hypothesis using live cell imaging on a DC sublayer and detected slightly slower movement and shorter tracks of cells expressing glycosylated FL BCRs as compared to control cells. Together, our results ascribe a role of the acquired glycosylation sites in FL BCRs for B-cell/DC interaction, thereby keeping the cells in the appropriate environment in a process that involves active signal transduction rather than triggering a classical antigen-induced BCR stimulation.