Strong Cell Surface Expression of the Toll-Like Receptor Homolog CD180 Identifies Circulating Cells of Marginal Zone Lymphoma From Other B-Cell Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1542-1542
Author(s):  
Laurent Miguet ◽  
Lucile Baseggio ◽  
Sarah Lennon ◽  
Pascale Felman ◽  
Luc Fornecker ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Marginal Zone ◽  
Extreme Values ◽  
Marginal Zone Lymphoma ◽  
Toll Like Receptor ◽  
Box Plot

Abstract Abstract 1542 Despite typical features described for marginal zone lymphoma (MZL), the distinction of MZL from other small mature B-cell leukemias/lymphomas (in particular atypical chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) or lymphoplasmacytic lymphoma (LPL) may be still difficult due to the lack of immunological positive markers of MZL and before complementary analysis (histology, karyotype). In order to find new markers, we conducted proteomic analyses on plasma membrane microparticles derived from malignant cells and identified the Toll-like receptor homolog CD180 protein as a candidate marker of MZL. This protein, also called RP105, is a leucin-rich repeat type 1 membrane protein with high extra-cellular homology to the LPS receptor. The relevance of this marker was evaluated in peripheral blood B-cells from 25 normal controls and 91 patients (MZL, n=30; CLL, n=30; MCL, n=16 and LPL, n=15) by flow cytometry. As already described by Porakishvili et al, normal B-cells displayed higher CD180 expression than CLL B-cells (MFI=5940+/− 1708 and MFI=150 +/− 794.4, respectively p<0.001, Mann-Whitney test). We observed also a low CD180 expression in MCL (1292 +/− 1084, p<0.001) and LPL (1443+/− 905.5, p<0.001), not different from CLL and significantly lower than control B-cells (see figure). On the contrary, staining of MZL was not different from controls (7856 +/− 1030, p>0.05), but significantly higher than CLL, MCL and LPL. In addition, among the lymphomas derived from MZ B-cell, CD180 expression was statistically higher in splenic diffuse red pulp lymphoma (SDRPL) than MZL. Intra-cellular staining of CD180 showed a significant positivity in tumoral B-cell tested (CLL, MCL, MZL), suggesting that the protein is produced but its expression at the cell surface is impaired. The reasons of this alteration are not known. Furthermore, cross-linking of this receptor induced a stronger increase of CD86 expression at the cell surface in MZL than in control B-cells and in CLL B-cells, suggesting an activated NF-kB pathway. These results strongly suggest that CD180 may be the first positive immunological marker for MZL, able to distinguish MZL from CLL, MCL, LPL in blood samples. Large prospective studies are needed to precise its diagnosis impact and its expression in the different subtypes of MZL. Figure: CD180 expression of normal and malignant B-cells. The box plot represents the median of mean fluorescence intensity (MFI), 25/75 percentiles and extreme values. Figure:. CD180 expression of normal and malignant B-cells. The box plot represents the median of mean fluorescence intensity (MFI), 25/75 percentiles and extreme values. Disclosures: No relevant conflicts of interest to declare.

Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2397-2397
Author(s):  
Gabriel Brisou ◽  
Laurent Jallades ◽  
Alexandra Traverse-Glehen ◽  
Francoise Berger ◽  
Aurélie Verney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cytidine Deaminase ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Marginal Zone B Cells ◽  
Activation Induced Cytidine Deaminase ◽  
Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

Preferential Acquision of N-Glycosylation Sites in the VDJ Region in Germinal Center B-Cell-Like Difusse Large B-Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1589-1589 ◽  
Author(s):  
Miguel Alcoceba ◽  
Elena Sebastián ◽  
Ana Balanzategui ◽  
Luis Marín ◽  
Santiago Montes-Moreno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Marginal Zone Lymphoma ◽  
Large B Cell Lymphoma ◽  
Glycosylation Sites ◽  
Large B Cell

Abstract Abstract 1589 Introduction: Acquired potentially N-glycosylation sites are produced by somatic hypermutation (SHM) in the immunoglobulin (Ig) variable region. This phenomenon is produced in ∼9% of normal B-cells and seems to be related to certain B-cell lymphoproliferative disorders (B-LPDs) such as follicular lymphoma (FL, 79%), endemic Burkitt lymphoma (BL, 82%) and diffuse large B-cell lymphoma (DLBCL, 41%). These data suggest that new potential N-glycosylation sites could be related to germinal center B (GCB)-LPDs. By contrast, in other B-LPDs, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), MALT lymphoma, Waldenström macroglobulinemia (WM) or multiple myeloma (MM), these modifications have not been analyzed in deep. Aims: To evaluate the acquisition of potential N-glycosylation sites in B-LPDs, including immunohystochemical DLBCL subtypes (GCB and non-GCB) and specific non-GCB-LPDs, such as hairy cell leukemia (HCL), splenic marginal-zone lymphoma (SMZL), CLL, MCL, ocular extranodal marginal zone lymphoma (OAEMZL), MM and WM. Patients: A total of 953 sequences (203 from our group and 750 previously published sequences) of B-LPDs were included. Diagnosis distribution was as follows: DLBCL (n=235), MCL (n=235), CLL (n=166), MM (n=96), OAEMZL (n=82), SMZL (n=68), WM (n=38) and HCL (n=33). Methods: Acquired N-glycosylation sites were counted according to the sequence Asn-X-Ser/Thr, where X could be any amino acid except Pro. Natural motifs in germline sequences of IGHV1–08, IGHV4–34 e IGHV-5a were not considered. Fisher test was used to perform comparisons between groups. To distinguish DLBCL biological subtypes (GCB and non-GCB DLBCL), Hans' algorithm was used. Results: A total of 83 out of the 235 DLBCL cases acquired at least a new N-glycosylation site, a higher value than in normal B-cells (35% vs. 9%, p<0.0001). Higher incidence of these motifs in the group of GCB as compared to non-GCB DLBCL were observed (52% vs. 20%, p<0.0001). Those cases diagnosed of HCL, CLL, MCL, MM, WM, OAEMZL and SMZL presented a reduced number of new N-glycosylation sites, showing similar values than normal B-cells (range 3–18%, p=ns). Conclusions: We described for the first time the pattern of N-glycosylation in HCL, SMZL, OAEMZL and in the immunohystochemical DLBCL subtypes, where the GCB-DLBCL showed a higher number of new N-glycosylation sites with respect to non-GCB DLBCL and other non-GCB-LPDs. The presence of novel N-glycosylation sites in FL, BL and in GCB-DLBCL strongly suggests that these motifs are characteristic of the germinal center B-LPDs. Disclosures: No relevant conflicts of interest to declare.

Identifying Phenotypically and Clinically Distinct Entities within the Spectrum of Marginal Zone Lymphoma and Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2652-2652
Author(s):  
Ruth M de Tute ◽  
Eve Roman ◽  
Andrew Jack ◽  
Roger G Owen ◽  
Andy Rawstron
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Clinical Features ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Lymphoplasmacytic Lymphoma ◽  
Normal Bone

Abstract Abstract 2652 The mature B-cell neoplasms marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LPL) and Waldenstrom macroglobulinemia (WM) display considerable overlap in clinic presentation, morphology, immunophenotype and genetic features. There is known to be poor reproducibility in the classification of these disorders and often only minimal diagnostic material is available. Multiparameter flow cytometry is a valuable tool for diagnosis and monitoring of B-cell disorders that can be applied to low cellularity samples or when the neoplastic cells represent a minority of total leucocytes. However, even when using large numbers of markers it is not possible to discriminate between MZL, LPL and WM and the aberrant expression of certain markers appears to occur with similar frequency in the different disease categories. The aim of this study was to determine whether there are phenotypically distinct entities within the spectrum of marginal zone and lymphoplasmacytic lymphomas, and to correlate these entities with clinical features. Samples from 146 people with a diagnosis of MZL, LPL or WM (122 bone marrow, 13 peripheral blood, 10 tissue biopsies), 8 with hairy cell leukemia, 10 reactive lymph node biopsies and 10 normal bone marrow samples were analysed using a panel of 39 markers. A minimum of 50,000 cells were acquired and analysed using a BD FACSCanto II with Diva software. Median fluorescence intensities were recorded for neoplastic CD19+ B-cell populations as well as for germinal centre (GC) and non-GC lymph node B-cells, B-progenitors, mature B-cells and plasma cell populations. Unsupervised cluster analysis was performed using dChip software. Clinical features and outcome data were collected and recorded centrally (www.hmrn.org.uk). Cluster analysis identified 5 entities with significant differences in their immunophenotypic profile. Neoplastic cells in all the MZL/LPL/WM groups typically expressed CD31, CD39 and CD49d with lack of CD10, CD23 and weaker expression of LAIR1 (CD305), CXCR5 (CD185) and CD22 compared to normal B-cells. CD5 and CD43 were expressed in a subset of cases but co-expression occurred in <1% of cases. The groups are characterised as follows: (1) clustered with normal mature B-cells, characterised by weaker CD20/CD95 and stronger CXCR5 expression relative to other MZL/LPL/WM cases, shorter survival, anemia and lymphadenopathy, typically requiring therapeutic intervention at diagnosis; (2) clustered with HCL, characterised by CD25 and CD11c expression and a higher degree of extranodal involvement; (3) did not cluster with other B-cell types, characterised by very strong CD79b and surface IgM expression often with anemia requiring supportive care; (4) characterised by strong CD24 expression with relatively good clinical course; and (5) clustered with normal bone marrow plasma cells, characterised by weak CD20, CD24 and IgM expression and also with better clinical features.Group2 year survivalAnemia (Hb <10.5g/dL)Lympho-cytosis (>4 × 10^9/L)Raised B2M (>2.7mg/L)Lymph-adenopathy (≥2 nodes)Extranodal involvementTherapeutic intervention at diagnosis115/24 (63%)10/19 (53%)9/19 (47%)8/10 (80%)12/19 (63%)4/19 (21%)14/19 (74%)228/37 (76%)8/25 (32%)11/25 (44%)6/11 (55%)9/23 (39%)10/23 (43%)9/25 (36%)325/31 (81%)14/24 (58%)5/23 (22%)11/18 (61%)4/19 (21%)5/19 (26%)13/24 (54%)422/27 (81%)5/22 (23%)5/22 (23%)10/17 (59%)5/20 (25%)4/20 (20%)8/22 (36%)522/26 (85%)5/16 (31%)5/16 (31%)6/8 (75%)8/14 (57%)5/14 (36%)5/16 (31%) The results indicate that the current diagnostic categories of MZL and LPL/WM have a great degree of overlap and comprise a number of phenotypically distinct entities which also appear to have different clinical features and outcomes. Enhancing the classification is likely to facilitate diagnosis, monitoring and identification of the underlying features of these B-cell malignancies. Disclosures: Rawstron: BD Biosciences: Patents & Royalties.

CD5-Negative Monoclonal B-Cell Lymphocytosis (MBL) Is Detectable In 9% of Adults Aged Over 40 but Phenotypes Associated with Clinically Common Non-Hodgkin Lymphomas Are Infrequent (<2%)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2002-2002
Author(s):  
Andy C Rawstron ◽  
Chi Doughty ◽  
Ruth M de Tute ◽  
Fiona Bennett ◽  
Selina Denman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
General Population ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Peripheral Blood ◽  
Marginal Zone ◽  
Residual Disease ◽  
High Sensitivity ◽  
Marginal Zone Lymphoma

Abstract Abstract 2002 Introduction: Monoclonal B-cells with a Chronic Lymphocytic Leukemia (CLL) phenotype are detectable in more than 10% of adults in the general population using high sensitivity flow cytometry assays designed to detect minimal residual disease after treatment. However, the prevalence of MBL with a phenotype corresponding to other B-lymphoproliferative disorders (B-LPD) is less than 2% of the general population even using the most sensitive assays. No studies have reported CD10+ MBL whereas several studies have demonstrated that the t(14;18) is frequently detectable in the general population at a level which should be detectable by flow cytometry. The lack of CD10+ MBL may indicate that the t(14;18) alone rarely results in the expansion of a clonal B-cell population in the blood, or that currently available assays are inadequate for detecting circulating follicular lymphoma. We have previously investigated 66 markers to determine the best candidates for diagnosis and monitoring B-LPD, which were then tested in over 1500 cases. We have developed a single-tube assay to screen for residual disease that can detect lymphoma cells when they represent as few as 1 in 10,000 leucocytes. The aim of this study was to asses the frequency with which lymphoma-phenotype monoclonal B-cells are detected in the general population using a high sensitivity assay. Methods: Cells from 679 individuals (342 male, 337 female, median age 64, range 40–99) with a normal blood count and no current or prior history of cancer were incubated with antibodies to Kappa, Lambda, CD19, CD20, CD5, CD10, LAIR1, CXCR5 and 0.5 million cells were acquired using a BD FACSCanto II cytometer. In cases with detectable MBL further phenotyping was performed and B-cells were selected and stored for FISH and molecular clonality studies. Results: MBL was detected 129/679 cases (19.0%): CLL-type MBL in 86/679 cases (12.7%), non-CLL MBL in 60/679 cases (8.8%) with both CLL-type and non-CLL MBL were present in 17/679 cases (2.5%). Within the non-CLL MBL group, in 21/60 cases the monoclonal B cells had no additional features to confirm a neoplastic population and it was not possible to ascertain whether these were neoplastic cells or a reactive population with a highly skewed kappa/lambda ratio. Of the remaining 39 specimens: none showed evidence of germinal centre differentiation; 12 (1.7% of total) showed a phenotype most consistent with marginal zone lymphoma/lymphoplasmacytic lymphoma; and 27 cases expressed strong levels of LAIR1 coupled with strong CD19 and CD20 and an extended phenotype that is relatively rare in clinical B-LPD, restricted to hairy cell leukemia and a small proportion (<15%) of marginal zone lymphomas. Conclusions: Using an assay designed for detection of residual disease in lymphoma it was possible to detect non-CLL phenotype MBL in 8.8% of individuals over 60 years of age with normal blood counts and no history of cancer. Only a small number of these cases had a phenotype comparable to follicular lymphoma or marginal zone lymphoma, which constitute the majority of clinically significant indolent B-LPD. The complete absence of cases with a germinal centre phenotype contrasts with the high frequency of detection of BCL2-IGH rearrangement by PCR in the peripheral blood of healthy individuals. The flow cytometry assay used in this study readily detects circulating cells with appropriate phenotype in patients known to have follicular lymphoma, diffuse large B-cell lymphoma or marginal zone lymphoma/lymphoplasmacytic lymphoma and the data indicate a high specificity for peripheral blood based diagnosis and monitoring in these conditions. The most clinically prevalent lymphomas are rarely detectable in peripheral blood in individuals without lymphadenopathy but there is a surprisingly frequent development in the general population of both CLL and a restricted subset of marginal zone lymphomas which may indicate a common developmental pathway. Disclosures: No relevant conflicts of interest to declare.

Peripheral Blood Disease Monitoring in Marginal Zone Lymphoma Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5307-5307
Author(s):  
Ruth de Tute ◽  
Andy C Rawstron ◽  
Roger G Owen ◽  
Andrew Jack
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Circulating Tumour Cells ◽  
Blood Samples ◽  
Flow Cytometric ◽  
Abnormal Cells

Abstract The use of sensitive flow cytometric techniques for the primary diagnosis and monitoring of patients with B-CLL is now well established. The aim of this study is to examine the feasibility of extending this approach to other indolent B-cell lymphoproliferative disorders. Such a technique, particularly if applicable to peripheral blood samples, would have significant benefits in allowing minimally invasive sequential monitoring of treatment responsiveness and early detection of relapse. Using a similar statistical approach to that used in the design of the CLL minimal disease flow cytometry panels we identified LAIR1, CCR6, CXCR5 and CD10 as markers that allowed discrimination between normal and neoplastic B-cells. These markers were incorporated into a 2 tube 8 colour assay together with CD19, CD5, CD20, CD22, CD45, kappa and lambda. Leucocytes were prepared using ammonium chloride lysis, incubated with these antibodies and washed before acquisition of 500,000 events on a FacsCanto flow cytometer (BD BioSciences, Oxford). As part of the evaluation of the technique we investigated peripheral blood samples from a total of 28 patients (14 male, median age 69.1 years and 14 female, median age 73.4 years), 22 with Marginal Zone Lymphoma/Waldenstrom’s Macroglobulinaemia (MZL) and a further 6 with Extranodal Marginal Zone Lymphoma (EMZL). Of the 22 MZL patients, 8 had active disease, 5 had stable disease that was untreated, 1 was on treatment and 8 were in remission. Of the extranodal MZL patients 1 had stable untreated disease, with 2 on treatment and 3 in remission. 77% (10/13) of MZL patients with active or stable untreated disease had detectable circulating tumour cells. Abnormal B-cells represented a median 2% of leucocytes (range 0.08 – 39%). The median absolute abnormal B-cell count was 0.34 ×109/l (range 0.003–9.6 ×109/l). The phenotypes of the cells detected showed a high level of heterogeneity. Abnormal cells were detectable in 22% (2/9) of cases on treatment or in remission, with a median % abnormal B-cells of 0.88% (range 0.35–5.37%) and a median absolute B-cell count of 0.09×109/l (range 0.02–0.386×109/l). None of the EMZL patients had abnormal cells detectable. Circulating tumour cells were detectable down to a level of 0.003×109/l and were found in a proportion of patients on treatment or considered to be in clinical remission. Methods used to monitor patients with indolent lymphoproliferative disorder need to take into account the phenotypic diversity found in marginal zone lymphoma compared to CLL. However the results demonstrate the feasibility of extending the use of flow cytometric residual disease assays to marginal zone lymphoma.

scholarly journals Detailed Functional Characterization of Splenic Marginal Zone Lymphoma: Uncovering Links between the Epigenetic and the Signaling Machinery

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1512-1512
Author(s):  
Stavroula Ntoufa ◽  
Maria Gounari ◽  
Nikos Papakonstantinou ◽  
Despoina Binou ◽  
Ioannis Tyritidis ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Hek293 Cells ◽  
Splenic Marginal Zone Lymphoma ◽  
Protein Levels ◽  
Stimulation Of

Splenic marginal-zone lymphoma (SMZL) ontogeny and evolution is a complex process, involving, amongst others, (super)antigenic stimulation, molecular deregulation of genes involved in the physiological differentiation of splenic marginal zone B cells (e.g. NOTCH2 and KLF2), and epigenetic alterations leading to silencing of different tumor suppressor genes and overexpression of oncogenes, including the PRC2-complex (EZH2, EED, and SUZ12). However, despite important recent progress, advances in the fundamental understanding of SMZL have not yet been translated in improved patient management, highlighting the need for further molecular investigations. In this context, here we aimed at detailed characterization of: (i) epigenetic modifiers, particularly the histone methyltransferase EZH2, a marker of clinical aggressiveness and druggable target in lymphoma; (ii) immune signaling capacity of SMZL B cells; and, (iii) antigen reactivity profile of the SMZL clonotypic B Cell Receptor immunoglobulin (BcR IG). Quantification of EZH2 mRNA levels using quantitative Real-time PCR (n=26 SMZL patients) and protein levels using western blotting (WB) (n=14) revealed that EZH2 was expressed in all cases yet in a heterogeneous manner (2^-ΔCt range: 0.079-1.38; ΕΖΗ2+ cells: 3.11%-44.8%). Interestingly, higher proliferation rates (Ki67+ cells) were observed in cases with high EZH2 protein levels (n=4) compared to cases with low EZH2 (n=6) (FD=2.6; p&lt;0.05). Stimulation of SMZL CD19+ B cells (n=8) through i) the BcR with anti-IgM ii) Toll-Like receptor 9 (TLR9) with CpG and iii) BcR/TLR for 60 minutes affected pBTK, pERK and pNF-kB levels as revealed by WB and flow cytometry (FCM). Although, great heterogeneity was observed in the responses between different cases, categorization of SMZL cases based on EZH2 levels showed that, at basal level, EZH2high cases expressed higher pBTK, pERK and pNF-κB compared to EZH2low cases (FC=2.4, FC=6,1, FC=12.7, respectively; p&lt;0.05 for all comparisons). BcR and TLR9 activation and, especially, co-stimulation with anti-IgM/CpG induced the expression of pBTK, pERK and pNF-κB in EZH2low cases, while the opposite was observed in EZH2high cases. Moreover, stimulation through both the BcR and BcR/TLR9 for 24 hours could also induce EZH2 expression compared to the unstimulated control cells (FC=1.6; p&lt;0.05 for both comparisons). To gain insight into the type of antigenic stimuli that could activate BcR signaling through the BcR we investigated the antigen binding profile of 10 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (mAbs). Cases were representative of the most common IGHV genes in SMZL (IGHV1-2*04, IGHV4-34 and IGHV3-23). Reactivity (at 15 μg/ml) against various autoantigens was evaluated by ELISA. Five of 9 (56%), 4/7 (57%), 5/10 (50%), 2/9(22%), 2/9 (22%), 3/9 (33%), 3/8 (38%), 4/8 (50%) and 4 of 9 (44%) SMZL IgM mAbs displayed reactivity against native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab')2 and the hapten trinitrophenyl respectively, with optical density (OD) values greater than the cut-off point. Moreover, 5/10 (50%) SMZL IgM mAbs recognized more than 2 antigens and were characterized as polyreactive. Each of these SMZL mAbs exhibited a distinct antigen polyreactivity profile, while heterogeneity was observed even amongst cases expressing the same gene or cases with similar IGHV gene mutational load. Recognition of epitopes on the surface of viable MEC1 B and HEK293 cells was assessed by FCΜ. Two of 3 and 5/11 mAbs bound to viable MEC1 B cells and HEK293 cells, respectively: binding to HEK293 cells was more pronounced for minimally mutated/unmutated versus mutated mAbs (p&lt;0.05). Importantly, stimulation of SMZL CD19+ B cells with the SMZL BcR cognate antigens actin and myosin resulted in regulation of pERK/pPLCγ2 in 3/6 and 1/2 cases, respectively, as revealed by WB. Overall, SMZL cells can be activated by microenvironmental signals albeit in a different manner depending on EZH2 levels, highlighting links between the epigenetic and the signaling machinery with potential therapeutic implications. Moreover, the recognition of a wide range of autoantigens by the SMZL mAbs indicates that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

2009 ◽  
Vol 417 (3) ◽  
pp. 673-683 ◽  
Author(s):  
Munetoyo Toda ◽  
Risa Hisano ◽  
Hajime Yurugi ◽  
Kaoru Akita ◽  
Kouji Maruyama ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Signalling ◽  
Negative Regulator ◽  
Mammary Adenocarcinoma ◽  
Marginal Zone B Cells ◽  
Tumour Bearing

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

scholarly journals Notch2-mediated plasticity between marginal zone and follicular B cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Markus Lechner ◽  
Thomas Engleitner ◽  
Tea Babushku ◽  
Marc Schmidt-Supprian ◽  
Roland Rad ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Close Contact ◽  
Immunological Function ◽  
Transitional B Cells ◽  
Bona Fide ◽  
Fate Decision ◽  
Follicular B Cells ◽  
Cell Follicle

AbstractFollicular B (FoB) and marginal zone B (MZB) cells are functionally and spatially distinct mature B cell populations in the spleen, originating from a Notch2-dependent fate decision after splenic influx of immature transitional B cells. In the B cell follicle, a Notch2-signal is provided by DLL-1-expressing fibroblasts. However, it is unclear whether FoB cells, which are in close contact with these DLL-1 expressing fibroblasts, can also differentiate to MZB cells if they receive a Notch2-signal. Here, we show induced Notch2IC-expression in FoB cells re-programs mature FoB cells into bona fide MZB cells as is evident from the surface phenotype, localization, immunological function and transcriptome of these cells. Furthermore, the lineage conversion from FoB to MZB cells occurs in immunocompetent wildtype mice. These findings demonstrate plasticity between mature FoB and MZB cells that can be driven by a singular signaling event, the activation of Notch2.

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