FALLAS BIFÁSICAS (FASE-A-FASE) EN EL DEVANADO DEL ESTATOR

Binding studies using oligomannosylated FL sIg and mannose-binding lectin (MBL), confirmed that glycans were accessible for binding to calcium-dependent (C-type) lectins (McCann et al., 2008), suggesting a novel interaction between tumour cells and

66 their microenvironment through oligomannose engagement. C-type lectins are pattern recognition receptors (PRR) that bind to glycans through their carbohydrate binding domains (2009). They have a wide variety of roles including phagocytosis/antigen presentation of pathogens and mediation of endogenous cell-cell interaction during the immune response (Dambuza and Brown, 2015, Geijtenbeek and Gringhuis, 2009). As genes associated with macrophages and dendritic cells are predictive of poor prognosis (Dave et al., 2004), it was hypothesised that lectins expressed on these immune cells were candidate molecules for interaction with sIg mannoses. Two major C-type lectins were proposed which have specificity for high mannose structures and are expressed by macrophages and dendritic cells; mannose receptor (MR) and DC-SIGN (DC-specific intercellular adhesion molecule-3–grabbing non-integrin). In vitro studies revealed that mannosylated sIgM from primary FL cells interacted with both lectins and induced sIgM- associated intracellular calcium fluxes, highlighting a functional interaction (Coelho et al., 2010). In addition, recombinant human DC-SIGN incubation with IgM positive FL cells led to phosphorylation of BCR signalling kinases, including ERK1/2 and AKT, and increased expression of MYC (Linley et al., 2015, Amin et al., 2015). This highlights the activation of the BCR pathway (and its associated targets) through an antigen- independent mechanism. Although phosphorylation of kinases was delayed and reduced in comparison to control anti-IgM treatment, the duration of response was longer (Linley et al., 2015). Furthermore, the sIg failed to undergo endocytosis, suggesting that constitutive activation of the BCR signalling pathway through DC-SIGN engagement is enough to maintain FL survival. These effects were lost with the treatment of Btk and Syk inhibitors, providing support to BCR activation via the lectin- mannose interaction (Linley et al., 2015). The proposed mechanism is summarised in Figure 1.9. DC-SIGN was found to be strongly expressed on M2 macrophages and dendritic cells derived from FL samples (Amin et al., 2015) which may have relevance to the poor prognosis associated with a high CD68+ macrophage count (Dave et al., 2004). It was also found to be in direct contact with FL cells in situ, indicating the feasibility of DC-SIGN as a FL specific BCR ligand within the disease niche (Amin et al., 2015). Bacterial lectins Pseudomonas aeruginosa and Burkholderia cenocepacia were also shown to bind to FL sIg and induce sIg-associated intracellular calcium fluxes (Schneider et al., 2015), indicating the vast array of potential lectin candidates that may be utilised in disease

67 pathogenesis. There is a dispute regarding the Ig isotype able to bind DC-SIGN. Linley et

al found both IgM+ and IgG+ FL cell were able to bind to DC-SIGN and activate signalling

(Linley et al., 2015). However, Amin et al found that IgG+ FL cells were poorly activated by DC-SIGN (Amin et al., 2015). These discrepancies may be due to technical and IgG+/IgM+ case variation between the two studies. However, as IgG+ FLs are more commonly self-reactive (Sachen et al., 2012), the lowered affinity to lectins may be due to alternative stimulation (via autoantigen) of the BCR within this isotype subgroup. This warrants further investigation.

While DC-SIGN and MR are well described in innate immunity (Engering et al., 1997, Geijtenbeek et al., 2000), the evidence is accumulating regarding their role in tumour immunity. Cross-linking of MR on immature dendritic cells with anti-MR antibody was shown to activate DC maturation and induce the secretion of anti-inflammatory cytokines and Th2 attracting chemokines, leading to negative regulation of Th1 polarised responses (Chieppa et al., 2003). Interestingly, similar responses are seen when the receptor is engaged with cancer-derived glycoproteins. For example, ligation of MR with the ovarian cancer derived glycoprotein TAG-72 on TAMs lead to increased IL-10 production and decreased CCL3 (Allavena et al., 2010), the Th1 attracting chemokine. This immune dampening response is also seen when DC-SIGN is engaged with colon cancer derived glycoproteins, leading to an increased IL-10 expression (Nonaka et al., 2008). IL-10 production promotes polarisation of M2 macrophages (Allavena et al., 2010), the phenotype displayed by FL derived TAMs. MR and DC-SIGN are highly expressed on TAMs (Amin et al., 2015), suggesting that these lectins play a role in polarising the immune cells to create a pro tumour, immune suppressive microenvironment. This suggests another dimension to the lectin-mannose interaction in the support of tumourigenesis.

Mannose-lectin interactions motifs could enable FL cells to survive and accumulate mutations within the hostile GC through both stimulating the BCR and creating a tumour supportive microenvironment. Determining how N-gly motifs influence disease evolution could provide validation for targeting this tumour-microenvironment interaction. Promising evidence has been seen in a case study in which ISFN cells gathered from several lymph nodes of a 69 year old male contained a conserved N-gly

68 motif (NCS) in the CDR3 region of the IGHV, suggesting N-gly motifs can be acquired within malignant precursor cells, perhaps in parallel with the t(14:18) translocation (Kosmidis et al., 2017). This was supported in another case study of a 35 year old male in which clonally related ISFN and FL cells shared an acquired motif within the CDR2 region of the IGHV (Mamessier et al., 2015). Having a functional mannose-lectin interaction at this early stage in disease development may explain how precursor cells are able to survive in the hostile germinal centre without the need for high affinity BCRs enabling accumulation of genetic hits required for malignant transformation.

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Figure 1.9: Model highlighting the inferred interaction between FL cells and the microenvironment via the

mannose-lectin interaction. N-glycosylation motifs located in the variable region of the sIg on FL cells is an acceptor site for high mannose glycans. C type lectins including DC-SIGN are expressed on macrophages and dendritic cells. These selectively bind to the high mannose structures, leading to the organisation of the BCR and CD19 co-receptor signalling platforms and downstream phosphorylation of signalling kinases, including SYK, BTK, ERK and AKT (10). How this interaction may affect the partnering immune cell expressing DC-SIGN/MR in the context of FL has not been investigated. Image adapted from Strout, 2015 (Strout, 2015).