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

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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ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽

10.1182/blood.v118.21.2844.2844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2844-2844

Author(s):

Noelia Purroy ◽

Eva Calpe ◽

Pau Abrisqueta ◽

Cecilia Carpio ◽

Carles Palacio ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

Tumor Growth ◽

B Cell ◽

Cell Lines ◽

Ectopic Expression ◽

Xenograft Model ◽

Lymphoid Organs ◽

Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

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Acquisition of Mannoses on the Surface Immunoglobulin Binding Site Reveals Functional Status and Cell of Origin in Diffuse Large B Cell Lymphomas

Blood ◽

10.1182/blood-2018-99-118318 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 677-677

Author(s):

Giorgia Chiodin ◽

Philip Rock ◽

Enrica Antonia Martino ◽

Beatriz Valle Argos ◽

Graham Packham ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Binding Site ◽

Tumor Cells ◽

Antigen Binding ◽

Cell Of Origin ◽

B Cell Lymphomas ◽

Antigen Binding Site ◽

Surface Immunoglobulin ◽

Large B Cell

Abstract Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown. While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010). In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars). We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into "DC-SIGN binders" (DB-DLBCL) vs "DC-SIGN non-binders" DLBCL (NB-DLBCL). Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites. We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC. Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by 'antigen'. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. Disclosures Packham: Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

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The BLyS Survival Pathway in NHL-B Cells: Constitutive NF-kB and NFAT Lead to Activation.

Blood ◽

10.1182/blood.v104.11.2279.2279 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2279-2279

Author(s):

Lingchen Fu ◽

Tamayo Archito ◽

Yen-Chiu Lin-Lee ◽

Lan Pham ◽

Linda Yoshimura ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Low Grade ◽

Rt Pcr ◽

Protein Levels ◽

Realtime Pcr ◽

Survival Pathway ◽

Hodgkin's Lymphomas

Abstract B-cell non-Hodgkin’s Lymphomas (NHL-B), neoplasms of the immune system have shown a significant increase in incidence in the USA over the last three decades. While the pathophysiology of the NHL-B is still unclear, the need to identify the relevant genes and critical signaling pathways, and their involvement in the disease processes in NHL-B have begun to be elucidated. Recently, B Lymphocyte Stimulator (BLyS) has been described as a relatively new member of the TNF ligand family, as a potent cell survival factor that is expressed in many hematopoietic cells, including neoplastic B cells. BLyS can bind to three receptors: TACI, BCMA, BAFF-R, and plays a critical role in B cell maturation, differentiation and proliferation. Relatively high levels of BLyS has been found in the serum of NHL-B patients as well as of the patients with autoimmune disease. The mechanisms of BLyS gene expression and regulation is still unclear, but we have recently found that BLyS is constitutively expressed in several NHL-B cell lines and patient tumor samples by RT-PCR, confocal microscopy, realtime PCR and flow cytometry (FCM). We detected high levels and differential expression of BLyS receptors (TACI, BCMA, BAFF-R) in several NHL-B cell lines by flow cytometry, RT-PCR and realtime PCR in both NHL-B cell lines and patient tumor samples. We have identified a single binding site for NF-kB and two binding sites for NFAT in the BLyS promoter. We also show in aggressive lymphoma B cells that constitutive NF-kB and NFAT binds to the BLyS promoter constitutively. Inhibiting NF-kB/NFAT activity levels, using the NF-kB inhibitors, BAY-11 or Velcade (PS-341), can decrease NF-kB binding activity in the BLyS promotor by EMSA. These inhibitors also decrease BLyS and BAFF-R mRNA and protein levels by realtime PCR and flow cytometry. Similarly, when NHL-B cells were transfected with dominant negative NFAT or NF-kB constructs, there is a 50% decrease in BLyS and BAFF-R expressions, demonstrating that both the ligand (BLyS) and the receptor (BAFF-R) expression are regulated by NFAT and NF-kB. Interestingly, follicular (low grade) lymphoma cells do not express constitutive NF-kB/NFAT activation, and barely detectable mRNA and protein levels of BlyS, but can be activated with exogenous CD154/anti IgM in vitro, activating NF-kB/NFAT and promoting binding to the BLyS promoter by EMSA. This results in a significant increase of BLyS protein level by flow cytometry. Our studies indicate that constitutive NF-kB and NFAT are critical transcriptional regulators of the BLyS survival pathway in malignant B cells that may provide a future therapeutic target in the aggressive NHL-B.

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Highly sensitive and unbiased approach for elucidating antibody repertoires

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608649113 ◽

2016 ◽

Vol 113 (28) ◽

pp. 7846-7851 ◽

Cited By ~ 31

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.

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“De novo Classification of Mouse B Cell Types using Surfaceome Proteotype Maps”

10.1101/620344 ◽

2019 ◽

Cited By ~ 2

Author(s):

Marc van Oostrum ◽

Maik Müller ◽

Fabian Klein ◽

Roland Bruderer ◽

Hui Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

De Novo ◽

Cell Types ◽

Glycosylation Sites ◽

Cell Subpopulations

AbstractSystem-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when sample is limiting. We miniaturized and automated the previously described Cell Surface Capture technology increasing sensitivity, reproducibility, and throughput. We used this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and used targeted flow cytometry to uncover developmental cell subpopulations.

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Differentiation-Stage-Specific Expression of MicroRNAs in B-Lymphocytes and Diffuse Large B-Cell Lymphomas (DLBCL)

Blood ◽

10.1182/blood.v112.11.805.805 ◽

2008 ◽

Vol 112 (11) ◽

pp. 805-805 ◽

Cited By ~ 3

Author(s):

Raquel Malumbres ◽

Robert Tibshirani ◽

Elena Cubedo ◽

Kristopher A Sarosiek ◽

Xiaoyu Jiang ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Memory B Cells ◽

Microrna Expression ◽

B Cell Differentiation ◽

Cell Of Origin ◽

Specific Expression ◽

Differentiation Stage

Abstract B-cell development and differentiation are complex processes controlled by distinct programs of transcriptional control. A large set of transcriptional factors together or in succession control this process and their deregulation may result in block of differentiation or malignant transformation. MicroRNAs are small RNAs that orchestrate cellular functions by modulating the level of their targeted proteins by either translational arrest or transcript degradation, and play a key role in cell differentiation, apoptosis, proliferation and cancer development. An increasing number of transcription factors are being found targeted by microRNAs. Emerging evidence suggests that differentiation stage-specific expression of microRNAs occurs in the hematopoietic system and during T cell differentiation. Only limited information exists on microRNA expression in normal B cell differentiation and its malignant counterparts. Herein we analyzed microRNA expression profiles in distinct peripheral B cell differentiation stages-naïve, germinal center (GC) centroblasts and memory cells as well as tonsilar T cells. Furthermore, microRNA profiling was performed in germinal center-like (GCB-like) and activated B-cell-like (ABC-like) DLBCL cell lines originating from distinct B-cell differentiation stages. RNA, extracted with mirVana kit (AMBION) from B cell subsets and T cells enriched from normal tonsils was hybridized on LC Sciences (Houston, TX) microarrays harboring 470 human microRNAs probes (Sanger miRBase Release 9.1). Expression of selected microRNAs was confirmed by ABI RT-PCR methodology. Unsupervised clustering of microRNAs with values present in at least 50% of the samples (122 probes) resulted in perfect differentiation-stage clustering of samples. Application of Statistical Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) methods (FDR= 10%) identified a 47 microRNA cell of origin classifier for B-cells differentiation stage; 27 of these microRNAs were upregulated and 20 downregulated in centroblasts compared to memory B-cells. MicroRNAs belonging to paralog microRNA clusters (e.g. miR17-92-1, miR363-106a and miR25-106b) demonstrated similar patterns of expression in specific differentiation stages. To identify specific microRNA targets, miRanda, TargetScan and PicTar programs were used. To experimentally confirm the targets, we assessed the effects of specific microRNAs on the expression levels of targeted proteins and on the luciferase reporter under the control of the wild type and mutated 3′ UTR regions of putative target genes. Using this experimental approach we identified lymphocyte-stage-specific microRNAs which expression inversely correlated and might regulate the expression of LMO2, BLIMP1 and IRF4 proteins distinctively expressed at different differentiation stages of B lymphocytes. For example, miR223, which expression is low in GC cells but is high in naïve and memory B cells, downregulates the expression of LMO2. We next analyzed microRNA expression in DLBCL cell lines. Clustering analysis, using the 47 microRNA cell of origin classifier perfectly classified GCB-like and ABC-like cell lines. Interestingly, the expression of microRNAs in both GCB-like and ABC-like DLBCL cell lines was more similar to normal centroblasts than to memory B cells, suggesting that both may originate from distinct subpopulations of GC lymphocytes. The similarity of microRNA expression in cell lines to centroblasts was striking, with only 16 microRNAs (1 upregulated and 15 downregulated in cell lines) showing noticeable differences in levels of expression compared to normal cells. These microRNAs might be involved in the process of lymphoma transformation. SAM analysis aimed to differentiate GCB-like and ABC-like cell lines identified 11 microRNAs, only 3 of which were present in the cell of origin classifier. This observation suggests that there is also a difference in expression of microRNAs not directly related to the distinct cell of origin between the DLBCL subtypes. In summary, our results demonstrate that the microRNA profile changes during the GC reaction as well as during malignant transformation. Specific microRNAs can regulate key transcription factors controlling the processes of lymphocyte differentiation and transformation.

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

Blood ◽

10.1182/blood.v126.23.2685.2685 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2685-2685 ◽

Cited By ~ 1

Author(s):

Lorenz Thurner ◽

Sylvia Hartmann ◽

Klaus-Dieter Preuss ◽

Natalie Fadle ◽

Maria Kemele ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Variable Region ◽

Cell Receptor ◽

Healthy Controls ◽

Cell Of Origin ◽

Vh Genes

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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Classification of mouse B cell types using surfaceome proteotype maps

Nature Communications ◽

10.1038/s41467-019-13418-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Marc van Oostrum ◽

Maik Müller ◽

Fabian Klein ◽

Roland Bruderer ◽

Hui Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

Cell Types ◽

Glycosylation Sites ◽

Cell Subpopulations

AbstractSystem-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when samples are limited. Here, we miniaturize and automate the previously described Cell Surface Capture (CSC) technology, increasing sensitivity, reproducibility and throughput. We use this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and use targeted flow cytometry to uncover developmental cell subpopulations.

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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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The Functional Role of Acquired N-Linked Glycosylation Sites On Follicular Lymphoma B Cell Antigen Receptors.

Blood ◽

10.1182/blood.v120.21.2704.2704 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2704-2704

Author(s):

Dunja Schneider ◽

Hendrik Veelken ◽

Hassan Jumaa

Keyword(s):

Immune System ◽

B Cells ◽

B Cell ◽

Innate Immune System ◽

Germ Line ◽

Variable Region ◽

Innate Immune ◽

Variable Regions ◽

Glycosylation Sites

Abstract Abstract 2704 Follicular lymphoma (FL) is an indolent B-cell lymphoma characterized by apoptosis resistance due to overexpression of Bcl-2 as a consequence of the t(14;18) translocation, ongoing somatic hypermutation (SHM), and expression of B-cell receptors (BCR) with glycosylation of the antigen binding sites. Translocation and concomitant Bcl-2 overexpression can be found in healthy human blood B cells and is insufficient to drive lymphoma outgrowth in mouse models. Since most FL cells still express a surface B cell receptor (BCR) despite the disruption of one immunoglobulin heavy chain allele by the t(14;18) translocation, expression of an antigen receptor seems to be indispensable for FL development. Around 80% of FLs possess asparagine (N)-linked glycosylation sites (amino acid sequence: N-X-S/T) in their BCR variable regions that are not encoded in germ-line but are acquired through SHM. In contrast to germ-line-encoded glycosylation sites in the constant BCR region, where normal processing of the glycans results in termination on branched sugars like sialic acid, the variable region glycosylation sites carry mannose-terminating sugars. Recently, it has been shown that C-type lectins bind to and stimulate FL cells. Such lectins are normally expressed on cells of the innate immune system, e.g. dendritic cells (DCs), which also reside in close interaction with the transformed B cells in germinal centers. Importantly, previous studies point to an outstanding role of the tumor microenvironment in survival and proliferation of the FL cells. In this study, we demonstrate that the variable region glycosylation in FL BCRs contribute to stimulation of the cells as well as adhesion to cells of the innate immune system. The BCR from six FL and the appropriate glycosylation-defective controls in which the N-linked glycosylation sequons are removed by replacing the asparagine (N) residues with glutamine (Q) residues were expressed in the tko cellular reconstitution system. In tko cells, the BCR signaling cascade can be rendered functional at will through a tamoxifen-dependent mutant of the signal transducer SLP-65 (Meixlsperger et al., Immunity 2007; Dühren von Minden et al., Nature 2012). Tko cells expressing FL BCRs and their glycosylation-defective controls were tested for binding of a recombinant DC-SIGN/Fc fusion protein by flow cytometry. The mannosylated FL-derived BCR but not glycosylation-mutated receptors bound DC-SIGN. Stepwise mutation of individual glycosylation sites demonstrated variable contribution to the strength of lectin binding. Despite this specific binding to mannosylated FL BCRs, DC-SIGN/Fc failed to induce significant calcium mobilization of transduced tko cells. Crosslinking with anti-IgM, in contrast, led to a readily detectable BCR-mediated signal, thereby demonstrating functionality of the transduced BCR. To study the role of mannosylated FL receptors in interaction with their environment, we co-cultured cells expressing FL receptors containing or lacking N-linked glycans in the variable regions together with macrophages. Western blot analyses with a pan-phosphotyrosine antibody demonstrated higher global tyrosine phosphorylation in the lysates of cells expressing glycosylated receptors, thereby indicating a specific role for mannosylated V-regions in FL stimulation. Glycan-mediated interactions fulfill multiple important functions in the mammalian immune system including pathogen recognition and cell adhesion or trafficking. DC-SIGN serves as receptor for the uptake of mannosylated pathogens and contributes to cell-cell interaction by binding to the heavily glycosylated ICAM-2/3 (intracellular adhesion molecules-2/3). In the case of FL, it is therefore conceivable that DC-SIGN expressed on follicular DCs binds to the heavily mannosylated FL BCRs and serves thereby as adhesion molecule to keep the FL B cells within the follicular structure. We tested this hypothesis using live cell imaging on a DC sublayer and detected slightly slower movement and shorter tracks of cells expressing glycosylated FL BCRs as compared to control cells. Together, our results ascribe a role of the acquired glycosylation sites in FL BCRs for B-cell/DC interaction, thereby keeping the cells in the appropriate environment in a process that involves active signal transduction rather than triggering a classical antigen-induced BCR stimulation. Disclosures: No relevant conflicts of interest to declare.

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