LRPAP1 Is a Frequent B-Cell Receptor Antigen in Mantle Cell Lymphoma (MCL)

Abstract Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens. Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1. Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36) Supported by Wilhelm-Sander-Stiftung Disclosures No relevant conflicts of interest to declare.

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Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v120.21.2880.2880 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2880-2880

Author(s):

Martin Trepel ◽

Fabian Muller ◽

Mareike Frick ◽

Janina Rahlff ◽

Claudia Wehr ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Phage Display ◽

B Cell ◽

Conflicts Of Interest ◽

Specific Binding ◽

Variable Region ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Fab Fragment

Abstract Abstract 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions: These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures: No relevant conflicts of interest to declare.

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High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Blood ◽

10.1182/blood.v128.22.3022.3022 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3022-3022

Author(s):

Jonathan Scott Rink ◽

Sol Misener ◽

Osman Cen ◽

Shuo Yang ◽

Leo I. Gordon ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Density Lipoprotein ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Cholesterol Homeostasis ◽

B Cell Lymphomas ◽

Bcr Signaling

Abstract Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas. Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk. Results: The Burkitt's lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death. Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. Disclosures Thaxton: Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

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Unmutated Ig VH Genes Are Associated With a More Aggressive Form of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v94.6.1848 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1848-1854 ◽

Cited By ~ 1826

Author(s):

Terry J. Hamblin ◽

Zadie Davis ◽

Anne Gardiner ◽

David G. Oscier ◽

Freda K. Stevenson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Somatic Mutation ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Cell Of Origin ◽

Trisomy 12 ◽

Vh Gene ◽

Vh Genes ◽

Variable Region Genes

Abstract Despite having several characteristics of naı̈ve B cells, chronic lymphocytic leukemia (CLL) cells have been shown in some cases to have somatically mutated Ig variable region genes, indicating that the cell of origin has passed through the germinal center. A previous study of patients with CLL found an association between lack of somatic mutation and trisomy 12 and, therefore, possibly with a less favorable prognosis. We have sequenced the Ig VH genes of the tumor cells of 84 patients with CLL and correlated our findings with clinical features. A total of 38 cases (45.2%) showed ≥ 98% sequence homology with the nearest germline VH gene; 46 cases (54.8%) showed >2% somatic mutation. Unmutated VH genes were significantly associated with V1-69 and D3-3 usage, with atypical morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Median survival for stage A patients with unmutated VH genes was 95 months compared with 293 months for patients whose tumors had mutated VHgenes (P = .0008). The simplest explanation is that CLL comprises 2 different diseases with different clinical courses. One, arising from a memory B cell, has a benign course, the other, arising from a naı̈ve B cell, is more malignant.

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ETS1 Phosphorylation at Threonine-38 Is a Marker of B Cell Receptor Activation, Associating with Cell of Origin and Outcome in Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.1755.1755 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1755-1755 ◽

Cited By ~ 1

Author(s):

Elaine Y Chung ◽

Maurilio Ponzoni ◽

Zijun Yidan Xu-Monette ◽

Valdemar Priebe ◽

Luciano Cascione ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Receptor ◽

B Cell Receptor ◽

Cell Of Origin ◽

Santa Cruz ◽

Large B Cell Lymphoma ◽

Type 3

Abstract Background.Diffuse large B cell lymphoma (DLBCL) represents the most common lymphoma subtype (30%-40% lymphomas). Gene expression profiling (GEP) identifies two main subtypes of DLBCL defined by their postulated cell of origin (COO). Activated B cell-like DLBCL (ABC DLBCL) is characterized by activation of B-cell receptor signaling and the nuclear factor kB pathway: these patients respond worse to standard R-CHOP but better to novel agents (ibrutinib or lenalidomide) than patients with DLBCL of the germinal-center-type (GCB DLBCL). We have previously reported an upregulation of the transcriptional factor ETS1 in up to 25% of DLBCL (Bonetti, Testoni et al. Blood, 2013). ETS1 is involved in many biologic processes, including B cell differentiation. ETS1 undergoes a series of post-translational modifications, and, in particular, ETS1 phosphorylation at Threonine 38 (p-ETS1) is a marker for ETS1 activation. Here, we present the impact of p-ETS1 in DLBCL cellular models and in a large series of clinical specimens. Patients and Methods. Levels of p-ETS1 (ab59179, Abcam) and, as controls, of ETS1 (C-20, Santa Cruz Biotechnology), ERK (93, Santa Cruz Biotechnology), p-ERK-Tyr204 (p-ERK; 7383, Santa Cruz Biotechnology) and IRF4 (4964, Cell Signaling Technology) were analyzed in cell lines derived from ABC (n.=7), GCB (n.=8) and type 3 (n.=4) DLBCL. p-ETS1 was examined by immunohistochemistry on clinical specimens: cases were defined as positive if > 10% of the neoplastic cells nuclei were p-ETS1 positive. Results.p-ETS1 was detected in ABC, not in GCB DLBCL cell lines (100% vs 0%, P<0.05), but also in 3/4 Type 3 (75%). All the cell lines expressed ETS1 and ERK. The ABC marker IRF4 was expressed in all ABC (100%), 1/4 type 3 (25%) and 0/8 GCB cell lines (0%). p-ERK was expressed in 6/7 ABC (86%), 2/4 Type 3 (50%) and 0/8 GCB (0%). Since ETS1 is a transcription factor and p-ETS1 is marker for its activation, we looked at the pattern of expression between nucleus and cytoplasm in five ABC DLBCL cell lines. p-ETS1 was present predominantly in the nucleus of all cell lines, while total ETS1 was in both cytoplasm and nucleus in 4/5, and only in the nucleus in 1/5 (TMD8). To evaluate the mechanisms sustaining p-ETS1, two ABC DLBCL cell lines (U2932, TMD8) were exposed to the PI3K-delta inhibitor idelalisib (1 μM), the BTK inhibitor ibrutinib (0.5 μM) and the MEK inhibitor pimasertib (0.5 μM) after stimulation or no stimulation with anti-IgM. In both cell lines, inhibition of BTK or MEK decreased the baseline levels of p-ETS1, while only inhibition of MEK was able to inhibit the increase in p-ETS1 induced by IgM stimulation. PI3K-delta inhibition only lead to a minimal reduction of the baseline p-ETS1 levels. Similar changes were seen for p-ERK. To understand the clinical significance of our findings, we assessed p-ETS1 expression in DLBCL biopsies. First, on a small series of 14 DLBCL cases classified for their COO using the Hans algorithm, p-ETS1 was mostly detected at nuclear level. The percentage of p-ETS1 positive cells was higher in ABC (no.= 7) than in non-ABC (no.= 7) (P 0.023), in agreement with the preclinical data. We then extended the analysis to a cohort of GEP-classified 315 de novo DLBCL cases treated with R-CHOP regimen collected as part of The International DLBCL Rituximab-CHOP Consortium Program Study (Xu-Monette et al, Blood 2013). The presence of p-ETS1 was further confirmed to be more frequent in ABC than in GCB cases: 79% (123/155) vs 57% (91/160) (P < 0.001). The p-ETS1 positive GCB DLBCL presented an inferior progression free survival (PFS) than p-ETS1 negative cases (P 0.034), while no association with outcome was detected in ABC DLBCL. Conclusions. Our data identify ETS1 phosphorylation at threonine 38 as i) associated with the ABC DLBCL phenotype; ii) associated with poor PFS in GCB DLBCL; iii) downstream to BCR signaling and amenable to pharmacological interventions with BTK and MEK inhibitors. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria.

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Splenic lymphoma with villous lymphocytes involves B cells with extensively mutated Ig heavy chain variable region genes

Blood ◽

10.1182/blood.v85.6.1603.bloodjournal8561603 ◽

1995 ◽

Vol 85 (6) ◽

pp. 1603-1607 ◽

Cited By ~ 60

Author(s):

D Zhu ◽

DG Oscier ◽

FK Stevenson

Keyword(s):

B Cells ◽

B Cell ◽

Heavy Chain ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Variable Region ◽

Low Grade ◽

Cell Of Origin ◽

Splenic Lymphoma ◽

Vh Genes

Splenic lymphoma with villous lymphocytes (SLVL) is a recently defined subgroup of chronic B-cell lymphoproliferative diseases. The characteristic morphology of the tumor cells, together with phenotypic and cytogenetic findings, indicate that it is a distinct entity, but the nature of the cell or origin and its relationship to other low- grade lymphomas is unclear. For B-cell tumors, analysis of the variable region heavy chain (VH) genes used to encode the clonal Ig has shown marked differences between histologic categories, both in gene usage and extent of somatic mutation. An investigation of VH genes used in five typical cases of SLVL has shown somatic hypermutation from germline sequences in all cases, indicating that the cell of origin has been exposed to the hypermutation mechanism. However, no clonal heterogeneity was detectable, demonstrating that the tumor cell does not accumulate further mutations. These characteristics are similar to those found in mature postfollicular B cells, such as plasma cells. The distribution of mutations leading to replacement amino acids differed among the cases, with three of five cases showing clear evidence for antigen selection.

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Unmutated Ig VH Genes Are Associated With a More Aggressive Form of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v94.6.1848.418k05_1848_1854 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1848-1854 ◽

Cited By ~ 23

Author(s):

Terry J. Hamblin ◽

Zadie Davis ◽

Anne Gardiner ◽

David G. Oscier ◽

Freda K. Stevenson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Somatic Mutation ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Cell Of Origin ◽

Trisomy 12 ◽

Vh Gene ◽

Vh Genes ◽

Variable Region Genes

Despite having several characteristics of naı̈ve B cells, chronic lymphocytic leukemia (CLL) cells have been shown in some cases to have somatically mutated Ig variable region genes, indicating that the cell of origin has passed through the germinal center. A previous study of patients with CLL found an association between lack of somatic mutation and trisomy 12 and, therefore, possibly with a less favorable prognosis. We have sequenced the Ig VH genes of the tumor cells of 84 patients with CLL and correlated our findings with clinical features. A total of 38 cases (45.2%) showed ≥ 98% sequence homology with the nearest germline VH gene; 46 cases (54.8%) showed >2% somatic mutation. Unmutated VH genes were significantly associated with V1-69 and D3-3 usage, with atypical morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Median survival for stage A patients with unmutated VH genes was 95 months compared with 293 months for patients whose tumors had mutated VHgenes (P = .0008). The simplest explanation is that CLL comprises 2 different diseases with different clinical courses. One, arising from a memory B cell, has a benign course, the other, arising from a naı̈ve B cell, is more malignant.

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VH and VL Genes in Hairy Cell Leukemia Reveal a Dynamic On-Going Modification of the Surface B-Cell Receptor.

Blood ◽

10.1182/blood.v106.11.287.287 ◽

2005 ◽

Vol 106 (11) ◽

pp. 287-287

Author(s):

Francesco Forconi ◽

T. Amato ◽

D. Raspadori ◽

S. S. Sahota ◽

M. A. Dell’Aversano ◽

...

Keyword(s):

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Hairy Cell ◽

Cell Of Origin ◽

Glycosylation Sites ◽

V Genes ◽

Vh Genes ◽

Low Levels ◽

A Minor

Abstract Immunoglobulin (Ig) gene analysis delineates critical features of the clonal history of a B-cell tumor. After antigen interaction, mature B-cells undergo somatic mutation of the V-genes and isotype switch recombination, generally in the germinal center (GC). Receptor revision by secondary recombination of the V-genes with re-expression of recombination activating gene (RAG) enzymes rarely occurs at this stage. From a small series, we have reported that most hairy cell leukemias (HCL) carry mutated VH genes, with low levels of intraclonal heterogeneity, while a minor subset have unmutated VH genes. Both subsets commonly have ongoing Ig isotype switch events and express activation induced cytidine deaminase (AID). However HCL lack the GC markers CD27 and CD38, and CD23, a chemokine essential for lymph node entry. In order to probe more fully the differentiation status of the cell of origin, both VH and VL tumor-derived genes were evaluated in an expanded series of 38 HCL. From analysis of VH, the VH3 family usage was most common (24/38, 63%), with significant preference of the VH3-30 member in 10/38 cases (26%, p=0,00001), and JH4b segment utilised in 50% of cases. Most HCL (35/38) carried variable tiers of mutations (87–98.6% homology to germline), with low level of intraclonal heterogeneity also in cases with <2% deviation from germline, while 3/38 (8%) displayed completely unmutated VH genes. Analysis of VL genes provided novel insights, when 21/38 HCL were evaluated. The λ light chain was most fequently used (13/21, 62%), to indicate preferential secondary rearrangement to λ chain. All λ cases used Jγ3 segment. Nineteen of 21 cases carried mutated VL genes (94,75%–99.6%) with low levels of intraclonal heterogeneity, while 2 cases carried completely unmutated VL genes, reflecting the status of the VH gene. Strikingly, in-frame functional secondary VL chain rearrangements were observed in the tumor cells of 2 of these cases (Vκ 1 and Vκ 2 in case R1, Vκ 1 and Vκ 2 in R2). Primary and secondary rearrangements showed mutations (98.1 and 99.6% homology in R1, 97.6 and 99.6% homology in R2). In both cases, RAG1 re-induction was identified by RT-PCR and sequence verification. Both cases expressed AID transcripts and displayed intraclonal mutations in the VH and/or the VL genes. These data suggest a dynamic, on-going modification of the B-cell receptor in tumor cells, including receptor revision, which occurs most likely in response to antigenic stimuli. N-glycosylation sites, commonly introduced in the V regions of tumors of the GC by somatic mutation, were not observed in the VH or in the VL functional genes, to support the concept that tumor events occur outside the GC. These data confirm heterogeneity in the cell of origin in terms of mutational status, with a minor subset with unmutated V-genes. Restricted V-gene segment usage, low levels of ongoing mutations with AID activated, and the new observation of receptor revision with re-expression of RAG enzymes indicate that selection by antigen could be a promoting factor in HCL development. Lack of novel glycosylation sites is in favour of interaction with antigenic stimuli occurring at extrafollicular sites.

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Mannosylation of the Tumor Immunoglobulin Variable Region Informs Cell of Origin and Environmental Interactions in DLBCL Subsets

Blood ◽

10.1182/blood-2019-127159 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1505-1505 ◽

Cited By ~ 1

Author(s):

Giorgia Chiodin ◽

Joel Allen ◽

Philip J Rock ◽

Enrica Antonia Martino ◽

Beatriz Valle Argos ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Variable Region ◽

Antigen Binding ◽

Cell Of Origin ◽

Glycosylation Sites ◽

Number Of Patients ◽

High Mannose

Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous diagnostic entity of B-cell tumors whose behavior is variably influenced by genetic changes and environmental stimuli. They are usually divided into two major subgroups, the germinal center B-cell-like DLBCL (GCB-DLBCL) and the activated B-cell-like DLBCL (ABC-DLBCL), with different cells of origin and distinct clinical behavior. From a previous analysis of a small number of patients, we found that a fraction of DLBCL acquires N-glycosylation sites by somatic hypermutation of the tumor surface immunoglobulin (sIg) variable region, suggesting a connection with follicular lymphoma (FL). In FL, this leads to addition of mannosylated glycans in the antigen binding site (sIg-Mann), which allow interaction with microenvironmental lectins including dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN). However, the distribution and the consequences of acquired N-glycosylation motifs in DLBCL was unknown. In this study, we investigated frequency, pattern and function of the mannosylated sIg in DLBCLs. DLBCL cell lines and primary samples were analyzed for the acquisition of the N-glycosylation motifs Asparagine-X-Serine/Threonine (N-x-S/T, where x ≠ Proline) in the tumor IGHV-D-J rearranged transcripts, and for binding to DC-SIGN by flow cytometry. HILIC-UPLC and crystallography were used to define structure of the glycans located in the sIg variable region. Interaction of sIg-Mann with DC-SIGN expressing cells was measured by flow cytometry and imaged by inverted fluorescence microscopy. Intracellular signaling was measured by Phosflow. Analysis of GCB-DLBCL and ABC-DLBCL cell lines and primary samples revealed that acquired N-glycosylation sites (AGS) were common in a subset of GCB-DLBCL (51%), especially in cases with a t(14;18) translocation (88%). Remarkably, the motifs were selectively acquired in the complementary-determining-regions (CDRs) of the tumor Ig (93%). In contrast, sites were infrequent in primary ABC-DLBCL (19%) and preferentially acquired in the framework regions (51% in all ABC-DLBCL cases, 88% in IGHV4-34 ABC-DLBCL). DLBCL cell lines with AGS which bound DC-SIGN had a t(14;18) translocation and were enriched with EZH2 and KMT2D mutations, while those without AGS, and unable to bind to DC-SIGN, were not. The sites acquired in the CDRs were permissive for addition of glycans terminating at high-mannose, as revealed by immunoblotting following EndoH treatment (that digests only glycans terminating at high-mannose) of the tumor sIg and by binding to soluble DC-SIGN. This was also confirmed by HILIC-UPLC and crystal structure of a sIg-Mann+ve lymphoma-derived recombinant Fab. Binding of DC-SIGN to sIg-Mann mediated an antigen-independent signal of lower levels than that mediated by anti-Ig, as measured by increased SYK phosphorylation in the tumor B cells. The sIg-Mann+ve GCB-DLBCL, but not sIg-Mann-ve DLBCL, formed clusters round DC-SIGN expressing cells. These interactions were inhibitable or disrupted by antibodies specifically targeting the DC-SIGN carbohydrate-recognition domain. Our results refine the phenotypic and functional characteristics of a GCB-DLBCL subset, in which the cell of origin has been selected to carry glycans terminating at high-mannose in the antigen-binding region. The acquisition of sites particularly in tumors harboring the t(14;18) translocation and mutations of epigenetic modifiers suggest a cell of origin common to FL, where these features occur early at transformation. Therefore, our data suggest the presence of a tumor cell ancestor with sIg glycans and genetic features common to FL and DLBCL. These results also document that those mannoses placed in the sIg variable region are functional and engage with DC-SIGN, to receive low level signals reminiscent of those protecting B cells from apoptosis. The possibility of inhibiting this antigen-independent interaction with anti-DC-SIGN antibodies in vitro suggests a potentially exploitable way for new therapeutic intervention. Disclosures Forconi: Menarini: Consultancy; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Honoraria; Gilead Sciences: Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau.

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Novel B-cell precursors blocked at the stage of DJH recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5216 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5216-5223 ◽

Cited By ~ 7

Author(s):

L Ramakrishnan ◽

N Rosenberg

Keyword(s):

B Cell ◽

Cell Lines ◽

Leukemia Virus ◽

Variable Region ◽

Immunoglobulin Gene ◽

Recombination System ◽

Rearrangement Process ◽

B Cell Precursors ◽

Enzymatic Machinery

Abelson murine leukemia virus-transformed cells have provided the principal model for study of the early events in immunoglobulin gene rearrangements. In this communication, we describe a new type of Abelson virus-transformed pre-B-cell line that is arrested at the DJH stage of the recombination process. These cells differ from other pre-B transformants with respect to two properties associated with the immunoglobulin rearrangement process. First, in contrast to cell lines undergoing VH-to-DJH joining in vitro, none of these cell lines contained detectable levels of RNAs transcribed from their unrearranged VH genes. Second, only some of the cell lines recombined exogenous heptamer-nonamer sequences, indicating that many of them have lost at least a portion of the enzymatic machinery that mediates recombination. The correlation between the absence of unrearranged VH RNAs and the inability to rearrange endogenous immunoglobulin gene segments suggests that VH gene transcription is required both to maintain an active recombination system and for the final step in variable-region formation.

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