DC-SIGN Binds Preferentially Highly Glycosylated IgM to Trigger Classical BCR Signaling in Follicular Lymphoma

BCR is supposed to deliver key stimulatory signals to malignant B cells in various mature hematological malignancies. In follicular lymphoma (FL), unlike in aggressive lymphomas, very few genetic alterations in BCR signaling pathway have been described, and BCR activation has been reported to rely on the dynamic interaction of mannosylated Ig with C-type lectins, such as Mannose receptor (MR) and DC-SIGN. In fact, the variable regions of FL Ig genes are characterized by the specific accumulation of N-glycan motifs introduced by the somatic hypermutation process and positively selected during lymphomagenesis. BCR activation is thus induced by an antigen-independent functional bridge between FL B cells and lectin-expressing non-malignant cells within tumor cell niche. In agreement, FL cells are well known to be strongly dependent on their microenvironment, including in particular stromal cells, CD4^pos T cells, and macrophages that collectively support malignant cell growth and suppress antitumor immune response. In particular, a high number of infiltrating tumor associated macrophages (TAM) is associated with poor prognosis in patients treated by conventional therapy. Since DC-SIGN and MR are expressed by myeloid cells, we explored the possibility that they could trigger FL BCR activation in vitro and in situ.

We first demonstrated that primary FL B cells of IgM isotype exhibited a higher response to anti-Ig triggering than normal germinal center B cells, as highlighted by phosphorylation of CD79a, Syk, BLNK, and Erk. Interestingly, IgG-expressing FL B cells showed reduced BCR activation compared to IgM-expressing malignant B cells, an observation that shed new light on the potential role of allelic paradox in FL, where malignant B cells essentially express membrane IgM despite active class-switch recombination on the translocated allele. Furthermore, only IgM-expressing FL cells were able to bind recombinant DC-SIGN with variable affinity, correlated to the level of glycosylation of Ig. Crosslinking of FL IgM by DC-SIGN induces an immune complex where CD19 was detected and led to a sustained phosphorylation of Syk, Akt, and Erk, whereas crosslinking by anti-IgM antibodies triggered a quick and transient activation. Co-culture of FL cells expressing highly glycosylated IgM with DC-SIGN^hi M2 macrophages, unlike with DC-SIGN^lo M1 macrophages, led to relocalization of DC-SIGN at the M2/B-cell interface and activation of Syk, Akt, and Erk in malignant B cells. Interestingly, M2-dependent activation of Erk was abrogated by BCR inhibitors such as imatinib. Finally, IL-4, which is overexpressed by infiltrating CD4^pos follicular helper T cells in FL, upregulated DC-SIGN expression on macrophages, thus favoring such BCR-dependent crosstalk.

Overall, our results support the hypothesis that glycosylated BCR contributes to DC-SIGN-dependent activation in FL and reveal the role of IgM isotype in this process. Such study paves the way for a better understanding of TAM/B cell crosstalk, which could constitute an important therapeutic target in this still fatal malignancy.