Freda Kathryn Stevenson

B cells expressing the IGHV4-34 gene sit at the center of a web of B-cell functions, ranging from natural antibodies induced by infection to autoimmunity and tumors. Tracking via the 9G4 monoclonal antibody (MoAb) specifically directed against the unique flag sequences in the first framework region 1 (FR1) of IGHV4-34, allows insight into the evolving nature and multiple functions of B cells expressing a single IGHV gene. IGHV4-34+ B cells are found early in development, and the expansion indicative of a superantigenic drive occurs with increasing age. IGHV4-34+ B cells secrete immunoglobulin M (IgM) in various hematologic conditions, including the mandatory association with cold agglutinins, and the transient expansions of usage (not always overt agglutination) are observed in Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumoniae, and other infections. Perhaps connected to that, certain autoimmune conditions, such as systemic lupus erythematosus, show increases in IGHV4-34 expression, which correlate with disease flares. Intriguingly, there is preferential usage of IGHV4-34 in a wide range of lymphomas and in the IgG variant of chronic lymphocytic leukemia, suggestive of an autoantigenic drive. Structurally, both the interaction of the 9G4 MoAb and the ability of the IGHV4-34–encoded protein to bind to N-acetyl lactosamine–containing sequences, rely on hydrophobic amino acids located in 2 positions of the looped FR1 sequence of the variable domain. This offers a focused site for blockade. IGHV4-34 is a paradigm of the plasticity of B cells, of interaction with potential autoantigens, and of their subversion in tumors.

Edited by Christian Steidl and Associate Editor Freda K. Stevenson, this Review Series provides state-of-the-art summaries on 4 integrated topics highlighting the importance of understanding the interactions between lymphoma cells and their tumor microenvironment. The emphasis is on follicular and diffuse large B-cell lymphomas, but the paradigms are relevant across the spectrum of B-cell lymphomas.

There are 2 mandatory features added sequentially en route to classical follicular lymphoma (FL): first, the t(14;18) translocation, which upregulates BCL2, and second, the introduction of sequence motifs into the antigen-binding sites of the B-cell receptor (BCR), to which oligomannose-type glycan is added. Further processing of the glycan is blocked by complementarity-determining region–specific steric hindrance, leading to exposure of mannosylated immunoglobulin (Ig) to the microenvironment. This allows for interaction with the local lectin, dendritic cell–specific ICAM-3–grabbing nonintegrin (DC-SIGN), expressed by tissue macrophages and follicular dendritic cells. The major function of DC-SIGN is to engage pathogens, but this is subverted by FL cells. DC-SIGN induces tumor-specific low-level BCR signaling in FL cells and promotes membrane changes with increased adhesion to VCAM-1 via proximal kinases and actin regulators but, in contrast to engagement by anti-Ig, avoids endocytosis and apoptosis. These interactions appear mandatory for the early development of FL, before the acquisition of other accelerating mutations. BCR-associated mannosylation can be found in a subset of germinal center B-cell–like diffuse large B-cell lymphoma with t(14;18), tracking these cases back to FL. This category was associated with more aggressive behavior: both FL and transformed cases and, potentially, a significant number of cases of Burkitt lymphoma, which also has sites for N-glycan addition, could benefit from antibody-mediated blockade of the interaction with DC-SIGN.

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with theMEG3lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature.GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of theGAB13′UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

Tumour cells from patients with chronic lymphocytic leukaemia (CLL) can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear to what extent these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied RNA sequencing, small RNA sequencing and DNA methylation array analysis to 38 CLL cases categorised into three groups by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. A significant proportion of the IGHV-associated miRNAs derived from chr14q32 clusters (14/38 (37%)), where all miRNAs were co-expressed with the MEG3 lncRNA, as part of the DLK1-DIO3 genomic imprinted region, a locus of known importance in the pathogenesis of other human tumours. Integrative in silico analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was predicted to be regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3’UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

Aim: T-helper cells could play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), a common B-cell neoplasm. Although CLL cells can present soluble antigens targeted from the B-cell receptor to T-helper cells via major histocompatibility complex (MHC) class II, antigens recognized by some CLL cells may be encountered in a particulate form. Here the ability of CLL cells to internalize and present anti-immunoglobulin M (IgM) beads as a model for the interaction of CLL cells with particulate antigens was investigated. Methods: The effect of anti-IgM beads on antigen presentation pathways was analyzed using RNA-seq and internalization of anti-IgM beads by primary CLL cells was investigated using confocal microscopy and flow cytometry. Antigen presentation was investigated by analyzing activation of a T-cell line expressing a T-cell receptor specific for a peptide derived from mouse κ light chains after incubating CLL cells with a mouse κ light chain-containing anti-IgM monoclonal antibody. Kinase inhibitors were used to characterize the pathways mediating internalization and antigen presentation. Results: Stimulation of surface IgM of CLL cells increased expression of the antigen presentation machinery and CLL cells were able to phagocytose anti-IgM beads. Internalization of anti-IgM beads was associated with MHC class II-restricted activation of cognate T-helper cells. Antigen presentation by CLL cells was dependent on activity of spleen tyrosine kinase (SYK) and phosphatidylinositol 3-kinase delta (PI3Kδ) but was unaffected by inhibitors of Bruton’s tyrosine kinase (BTK). Conclusions: CLL cells can internalize and present antigen from anti-IgM beads. This capacity of CLL cells may be particularly important for recruitment of T-cell help in vivo in response to particulate antigens.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Targeted therapies have been central to improvements in the treatment of hematologic malignancies. We present a review series edited by Andrew Roberts and Freda Stevenson focusing on Bruton tyrosine kinase inhibitors, phosphoinositide 3-kinase inhibitors, and BH3 mimetics and their role in the treatment of chronic lymphocytic leukemia, non-Hodgkin lymphomas and acute myeloid leukemia.

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 Ca²⁺ 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.