In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are ...tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell-cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca
response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.
The B-cell receptor (BCR) is essential to the behavior of the majority of normal and neoplastic mature B cells. The identification in 1999 of the two major CLL subsets expressing unmutated ...immunoglobulin (Ig) variable region genes (U-IGHV, U-CLL) of pre-germinal center origin and poor prognosis, and mutated IGHV (M-CLL) of post-germinal center origin and good prognosis, ignited intensive investigations on structure and function of the tumor BCR. These investigations have provided fundamental insight into CLL biology and eventually the mechanistic rationale for the development of successful therapies targeting BCR signaling. U-CLL and M-CLL are characterized by variable low surface IgM (sIgM) expression and signaling capacity. Variability of sIgM can in part be explained by chronic engagement with (auto)antigen at tissue sites. However, other environmental elements, genetic changes, and epigenetic signatures also contribute to the sIgM variability. The variable levels have consequences on the behavior of CLL, which is in a state of anergy with an indolent clinical course when sIgM expression is low, or pushed towards proliferation and a more aggressive clinical course when sIgM expression is high. Efficacy of therapies that target BTK may also be affected by the variable sIgM levels and signaling and, in part, explain the development of resistance.
Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL ...marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.
Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and ...Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Because ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment, combined with dissecting functional interactions of Bcl-2 family members, in an in vitro model of venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than did cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed; significantly, the pretreatment profile reappeared in patients who relapsed on ibrutinib. However, in response to venetoclax, Bcl-2 members displayed an early increase, underlining the different modes of action of these 2 drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1, and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that proapoptotic Bim interacts with antiapoptotic Bcl-2 members in a sequential manner: Bcl-2 > Bcl-XL > Mcl-1 > Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than is Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.
The clue that surface Ig (sIg) is implicated in the pathogenesis of follicular lymphoma (FL) is that in FL the vast majority of the sIg variable regions are structurally modified by insertion of ...mannose residues into the antigen-binding sites. This is tumor-specific and reflects positive selection of sequence motifs for glycan addition introduced during somatic hypermutation. The termination at high mannoses is very unusual in cell surface molecules and it confers an ability to interact with local lectins expressed by macrophages. In this respect, the mannose cloaking of the sIg receptor resembles that of human immunodeficiency virus (HIV), which acquires a similar mannose coat to facilitate binding to macrophages.
The lectin DC-SIGN is upregulated in FL, likely via local IL-4, and is the strong lectin candidate. To address the question of the functional implications of interaction between DC-SIGN and sIgM we used a FL-derived cell line (WSU-FSCCL) which expresses sIg-mannoses. We found that two recombinant derivatives of DC-SIGN (DC-SIGN-Fc or DC-SIGN-HA) bind to the sIgM but not to a cell line which express sIgM without mannose insertion. Although the mannoses are an integral part of the sIgM, and anti-IgM induces a Ca2+ flux, DC-SIGN binds but does not mimic anti-IgM. In the WSU-FSCCL cell line it remains on the surface IgM without inducing a Ca2+ response and without mediating endocytosis of the sIgM.
However, DC-SIGN does act on the sIgM as revealed by the effects of pre-exposure of the cells to either DC-SIGN derivative, which, although there is no blocking of access of anti-IgM, alters the ability of the cell to respond to stimulation. Pre-exposure appears to partially paralyze the subsequent sIgM-induced Ca2+ flux indicating a lectin-mediated modification of sIgM function.
On engagement by anti-IgM, sIg undergoes conventional endocytosis and this was confirmed in WSU-FSCCL. Inhibitors which block signaling did not affect endocytosis, indicating bifurcation of signaling and endocytic pathways. Knowing that DC-SIGN derivatives, again in contrast to anti-IgM, did not induce endocytosis of sIgM, we then asked if the paralysis of the sIgM-mediated Ca2+ flux by pre-exposure to lectin affected anti-IgM-induced endocytosis, and found that it did not. This confirms the separation of signaling and endocytosis and indicates that whatever change is occurring in sIgM due to lectin exposure does not remove the sIgM from the endocytic machinery.
We then investigated primary FL cases, using a splenic FL and 2 lymph node FLs, all carrying N-glycosylation motifs and able to bind DC-SIGN. We focused on the DC-SIGN-HA derivative, which gives no detectable Ca2+ flux signal by itself. Binding of the lectin again paralyzed subsequent anti-IgM-induced Ca2+ flux in all cases (Fig.1A), confirming the results with the cell line. Further investigation revealed that the lectin prevents early events after antigen binding to the sIg receptor reducing SYK phosphorylation and leading to a reduced Ca2+ mobilization (Fig.1B).
In conclusion, modulation of the B-cell receptor by mannose addition to the antigen-binding site allows lectin access from innate microenvironmental cells. The effect of this is to provide a low level/null signal without loss by endocytosis. The novel finding is that this interaction then lowers the function of the B-cell receptor and perhaps blocks potential interference by antigen. There may be a parallelism with reports of modification of T-cell receptor function by galectins, with both pointing to the role of post-translational modification adding another layer of control on the operation of the major immune receptors. In the case of FL, this has been exploited to maintain tumor cells in the hostile environment of the germinal center. The apparently lymphoma-specific adaptation offers opportunities for targeted inhibition of the interaction.
Display omitted
Forconi:Janssen-Cilag: Consultancy; Abbvie: Consultancy. Packham:Aquinox: Research Funding.
In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated
and recover
, suggesting a reversible ...influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM.
We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL.
At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture
. The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway.
This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy.
.
PDF
