Abstract
The selective acquisition of oligomannose-type glycans at the antigen-binding site of the surface immunoglobulin (sIg-Mann) is a tumor-specific post-translational modification of all follicular lymphomas (FL). Our immunogenetic and structural analyses have revealed that these oligomannoses at N-glycosylation sites are universally acquired and persist during the entire natural history of FL from the early stages throughout transformation into EZB lymphoma (Chiodin G, Blood 2021). If a FL cell loses these sites, which is rare, the progeny disappears unless a new glycosylation site is acquired (Odabashian M, Blood 2020). These data indicate that sIg-Mann is a fundamental functional requirement for the entire clonal population of all FLs. The low-affinity interaction of sIg-Mann with its specific ligand dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), expressed on interfollicular M2-macrophages and follicular dendritic cells (FDC) of FL (RadtkeA, BioRxiv, 2022), distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via AKT and MYC and not endocytosis (Linley A, Blood 2015). However, the function of DC-SIGN:Ig-Mann remains to be further elucidated. In this study, we investigated the hypothesis that the specific engagement of DC-SIGN:Ig-Mann interaction functions to promote homing, while maintaining selective growth and survival signals. We found that DC-SIGN induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1 and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN with an anti-DC-SIGN antibody specific to the carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion, confirming that adhesion was mediated by the selective interaction with the oligomannose of sIg-Mann. Although intracellular signals were significantly lower than those by soluble anti-IgM F(ab)2 (here used to mimic the binding of cross-linking polyvalent antigen to Ig protein), the level of adhesion induced by DC-SIGN was similar to that induced by anti-IgM F(ab)2. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable by immunoblotting. However, either proximal inhibition of the PI3K/AKT pathway or distal inhibition of ARP2/3, formin, Rac and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. This suggested that Ig-Mann-mediated adhesion required both low-level intracellular signals and membrane adaptation (Valle-Argos, Sci Rep 2021). Remarkably, unlike anti-IgM F(ab)2, adhesion was specific to VCAM-1 but not to other stromal elements including fibronectin (FN). Instead, using the sIg-Mann+ve (IGHV4-34+ve) WSU-FSCCL cells, DC-SIGN appeared to mimic the interaction of monovalent ligands, including Fab fragments to either the Ig constant (anti-IgM Fab) or Ig variable region (9G4 Fab, which recognizes an epitope in the framework region 1 of IGHV4-34). Like DC-SIGN, Fab fragments induced selective adhesion to VCAM-1 and low-level signals via AKT, but neither endocytosis nor adhesion to FN. These results identify an important functional role of the DC-SIGN:sIg-Mann in mediating a signal that is tuned at the right threshold to promote selective and, in the absence of Ig endocytosis, persistent adhesion to environmental VCAM-1, determining homing of the FL tumor cells in the lymph node. This tumor-specific antigen-independent low-affinity DC:SIGN-Ig-Mann interaction contrasts to conventional high-affinity antigen:Ig protein interaction, which is prevented in FL (Schneider D, Blood 2015) and would instead lead to endocytosis and death. Therapeutic interruption of the interaction of DC-SIGN+ve cells with sIg-Mann+ve FL cells would provide a new way to displace the tumor cells from their prosurvival microenvironment.
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