Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior

Blood ◽  
2021 ◽  
Author(s):  
Giorgia Chiodin ◽  
Joel D. Allen ◽  
Dean Bryant ◽  
Philip Rock ◽  
Enrica Antonia Martino ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Variable Region ◽  
Cell Receptor ◽  
Antigen Binding ◽  
Post Translational Modification ◽  
Antigen Binding Site ◽  
Lymphoma Cells ◽  
X Ray Crystallography ◽  
Significant Gene

Glycosylation of the surface immunoglobulin 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 (DLBCL) which acquire N-glycosylation sites selectively in the immunoglobulin (Ig) complementary-determining-regions (CDR) of the antigen-binding sites. Mass-spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures due to burial in the CDR loops. Acquisition of sites occurs in ~50% of germinal center B-cell-like 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 appears not 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 associate with more rapid disease progression and with significant gene-set enrichment of the B-cell receptor, PI3K/AKT/MTORC1, 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 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 immunoglobulin is a post-translational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

scholarly journals Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

2006 ◽  
Vol 282 (10) ◽  
pp. 7405-7415 ◽  
Author(s):  
Catherine M. Radcliffe ◽  
James N. Arnold ◽  
David M. Suter ◽  
Mark R. Wormald ◽  
David J. Harvey ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Binding Site ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Lymphoma Cells

Immunogenetic Profiling of Follicular Lymphoma Reveals Universal N-Glycosylation Sites Introduced into the B Cell Receptor by Somatic Mutation and Suggests Relevance to Lymphoma Pathogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 606-606
Author(s):  
Christian H. Ottensmeier ◽  
Katy J. McCann ◽  
Peter Johnson ◽  
Freda K. Stevenoson
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutation ◽  
Essential Feature ◽  
Variable Region ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Cell Repertoire ◽  
Antigen Binding Site ◽  
Glycosylation Sites

Abstract Immunogenetic analysis of B-cell malignancies can provide important information that relates to the cellular origin and clonal history of these lymphomas and give clues as to possible pathogenic mechanisms. In follicular lymphoma (FL), immunoglobulin variable region (V) genes are commonly somatically mutated and display intraclonal heterogeneity consistent with location in the germinal centre (GC). In this analysis of 44 cases of FL we find that, with minor exceptions, both the VH and VL gene usage reflects that of the normal B cell repertoire, indicative of a common antigenic drive and in support of a final transforming event in the GC. We have previously reported a high incidence of potential N-glycosylation sites in the VH genes of FL, which have been introduced by the process of somatic mutation. Here we have assessed both the VH and VL genes and find that sites are universally present and further demonstrate that they are available for functional glycosylation. The majority of sites are found in VH (81%) and are located predominantly within CDR2 and CDR3, with few sites present in the FRs. Sites are also evident in VL (45%) where they are focused mainly in CDR3 and CDR1. A minor subset (10%) has sites in VL only. In total, 26 different N-glycosylation motifs were observed, with NIS being the most commonly used. The natural motif in the V4–34 germline gene appears unimportant, and can be lost. Scrutiny of the somatic mutations giving rise to these motifs reveals that the acquisition of sites was predominantly (73%) achieved by a single amino acid (aa) replacement to Asn at position 1 of the motif, either with or without an additional, non-essential aa replacement at another position. Common ‘hotspots’ were observed within the CDR2 for the VH gene segments V3–23, V3–48, V3–07 and V3–15. It appears likely that the acquisition of N-glycosylation sites in the antigen-binding site during somatic mutation in the GC and the subsequent addition of oligosaccharides is important to the lifestyle of FL and may provide a critical second tumorigenic event. In turn, it may be possible to exploit this seemingly essential feature to develop novel therapeutic approaches.

scholarly journals Lymphoma-Specific Subversion of B-Cell Receptor Signaling By Macrophage Lectins

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2865-2865 ◽  
Author(s):  
Beatriz Valle Argos ◽  
Giorgia Chiodin ◽  
Francesco Forconi ◽  
Richard Burack ◽  
Philip Rock ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Antigen Binding ◽  
Sequence Motifs ◽  
Post Translational Modification ◽  
Antigen Binding Site ◽  
Variable Regions

Abstract 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. Figure 1. Figure 1. Disclosures Forconi: Janssen-Cilag: Consultancy; Abbvie: Consultancy. Packham:Aquinox: Research Funding.

Massive Parallel Sequencing of Full-Length B-Cell Receptor Sequences Reveals HLA-Dependent Shaping of the B-Cell Immune Repertoire

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4143-4143
Author(s):  
Marvyn T. Koning ◽  
Sander A.J. van der Zeeuw ◽  
Marcelo Navarrete ◽  
Cornelis A.M. van Bergen ◽  
Valeri Nteleah ◽  
...  
Keyword(s):  
T Cell ◽  
Follicular Lymphoma ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Full Length ◽  
Lymphoma Cells ◽  
Hla Alleles ◽  
Hla Binding

Abstract Peptides of the B-cell receptor (BCR) may be presented in HLA molecules and therefore be recognized as epitopes by T cells. Bioinformatic evidence indicates that follicular lymphoma cells are selected against expression of a clonal BCR with a high cumulative predicted binding of BCR-derived peptides to the respective patient's HLA complex (Strothmeyer, Blood 2010). This observation suggests T-cell-mediated immunosurveillance against outgrowth of follicular lymphoma cells according to BCR HLA binding strength. Here, we investigate whether this phenomenon pertains to peripheral B cells in 6 healthy donors: 2 donors homozygous for HLA A01*01 / B08*01, 2 homozygous for HLA A02*01 / B7*02, and 2 donors heterozygous for these alleles. Unbiased representation of full-length V(D)J sequences was considered essential for correct data interpretation. PCR primers annealing to conserved motifs of BCR variable regions (e.g. BIOMED-2 protocol) fail to amplify a fraction of BCR, particularly those modified by somatic hypermutation. Therefore, we developed an improved anchored PCR strategy: cDNA was synthesized from poly(A)-RNA from peripheral blood with primers that anneal to specific Ig constant regions. In the same reaction, the 3' cDNA end is extended by switching to an oligonucleotide template containing an anchor sequence (SMART technology; Clontech). Anchor-tagged cDNA was amplified with a primer annealing to the anchor in combination with a nested constant region-specific reverse primer. Dumbbell adapters were added to the termini of 250 ng of purified PCR products. Circular consensus sequencing of single molecules was performed on the PacBio platform (Pacific Biosciences). Using one SMRT PacBio cell per amplicon, separate sequence libraries were created for μ, γ, κ, and λ BCR transcripts. Sequences covered by at least five reads were selected with SMRT Portal software to obtain >95% of sequences without sequence errors as demonstrated on multiple B-cell lines. Selected sequences were analysed by HighV-QUEST software (Alamyar, Immunome Research 2012). After exclusion of non-BCR sequences and duplicate BCR transcripts, a median of 5318 (range: 670-8752) individual BCR sequences was obtained per library. Binding affinity of nonamers in in-silico-translated BCR were calculated for the 4 HLA alleles by the NetMHC 3.4 algorithm. The fractions of BCR lacking any weak HLA binding peptide (NetMHC IC50 <500nM) within a library were compared between donors positive or negative for any HLA molecule. μ VDJ transcripts without HLA binding peptides were significantly more frequent for all HLA alleles in donors that actually express that particular allele (Table). With the exception of HLA A01*01, similar results were observed for γ transcripts. While the fraction of κ VJ transcripts without an HLA binder was overall higher in HLA A01*01 and B08*01, HLA-positive individuals had higher proportions of non-HLA binding sequences. λ transcripts were less likely to contain HLA binders with respect to HLA B07*02 and B08*01 but not to the HLA A alleles. Analogous analyses were performed for CDR3 regions as annotated by HighV-QUEST plus six amino acids on either flank. In 10 of 16 analyses, CDR3 sequences were less likely to contain an HLA binder in HLA-positive individuals; in three analyses an opposite effect was seen (Table). These results indicate that the peripheral BCR repertoire is shaped by HLA alleles in healthy individuals, most likely by T-cell mediated recognition of BCR peptides. Ongoing studies expand this fundamental finding with respect to the IC50 threshold, the number of nonamers, and additional HLA alleles. Our results warrant investigation of the potential role of HLA-dependent shaping of the BCR repertoire for the immune defense and the development of autoimmune disease and B-cell lymphoma. Table 1V(D)J without HLA binding peptideCDR3 without HLA binding peptideHLADonorμγκλμγΚλ A01*01Positive21%41%61%37%87%90%98%70%Negative16%42%59%38%92%92%96%65%P<0.001n.s.<0.01n.s.<0.001n.s.<0.01<0.001 A02*01Positive6%4%3%32%77%77%77%70%Negative4%1%2%32%75%69%78%78%P<0.001<0.001<0.01n.s.<0.01<0.001n.s.<0.001 B07*02Positive31%13%3%13%79%73%91%96%Negative27%8%2%6%79%69%90%98%P<0.001<0.01<0.01<0.001n.s.<0.05<0.05<0.001 B08*01Positive30%35%64%64%89%87%92%96%Negative14%28%62%61%88%82%90%93%P<0.001<0.001<0.01<0.001<0.01<0.001<0.01<0.001 Disclosures No relevant conflicts of interest to declare.

scholarly journals Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 3135-3142 ◽  
Author(s):  
Jonathan M. Irish ◽  
Debra K. Czerwinski ◽  
Garry P. Nolan ◽  
Ronald Levy
Keyword(s):  
B Cells ◽  
Cell Signaling ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cells ◽  
B Lymphoma Cells ◽  
Bcr Signaling ◽  
B Cell Subsets

Abstract The B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (κ or λ) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.

scholarly journals Highly sensitive and unbiased approach for elucidating antibody repertoires

2016 ◽  
Vol 113 (28) ◽  
pp. 7846-7851 ◽  
Author(s):  
Sherry G. Lin ◽  
Zhaoqing Ba ◽  
Zhou Du ◽  
Yu Zhang ◽  
Jiazhi Hu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Variable Region ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Variable Regions ◽  
Antibody Repertoires ◽  
B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

scholarly journals IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution

Blood ◽  
2020 ◽  
Vol 135 (11) ◽  
pp. 834-844 ◽  
Author(s):  
Mariette Odabashian ◽  
Emanuela Carlotti ◽  
Shamzah Araf ◽  
Jessica Okosun ◽  
Filomena Spada ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Dna Sequences ◽  
Somatic Hypermutation ◽  
Variable Region ◽  
Cell Receptor ◽  
Tumor Evolution ◽  
Growth And Survival ◽  
Calcium Dependent ◽  
Glycosylation Sites

Abstract Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells’ dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2562-2568 ◽  
Author(s):  
Delin Zhu ◽  
Helen McCarthy ◽  
Christian H. Ottensmeier ◽  
Peter Johnson ◽  
Terry J. Hamblin ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutation ◽  
Germinal Center ◽  
Variable Region ◽  
B Cell Lymphoma ◽  
Single Chain ◽  
Single Chain Fv ◽  
Glycosylation Sites ◽  
Vh Genes

Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional VH genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P &lt; .001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv inPichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. VH sequences of nonfunctional VH genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.

scholarly journals The relationship between variable region determinants and antigen specificity on mitogen reactive B cell subsets.

1982 ◽  
Vol 156 (3) ◽  
pp. 924-929 ◽  
Author(s):  
D Primi ◽  
F Mami ◽  
C Le Guern ◽  
P A Cazenave
Keyword(s):  
B Cell ◽  
Variable Region ◽  
Antigen Specificity ◽  
Antigen Binding Site ◽  
V Region ◽  
Set Up ◽  
Cell Mitogen ◽  
B Cell Subsets ◽  
The Relationship ◽  
Beta Galactosidase

On the basis of previous frequency determinations we could set up large numbers of cultures, each containing less than one competent precursor B cell specific for beta-galactosidase or for each of three idiotopes previously found on a monoclonal anti-beta-galactosidase antibody. Cultures were polyclonally activated by either lipopolysaccharide or Nocardia-delipidated cell mitogen. Each culture supernatant was individually tested for hemagglutination activity against sheep erythrocytes coupled with beta-galactosidase or with each of the three purified monoclonal anti-idiotypic antibodies. The results showed that only a minority of those clones positive for only one or two idiotopes recognized antigen. However, all those clones simultaneously positive for the three V region determinants recognized beta-galactosidase. The implications of these results for our understanding of the relationship between the antigen-binding site and idiotope expression are discussed.

Sign in / Sign up

Export Citation Format

Share Document