The Immunoglobulin Gene Repertoire of Low-Count CLL-Like MBL Is Different from CLL: Diagnostic Considerations and Implications for Clinical Monitoring

Abstract The term Monoclonal B Lymphocytosis (MBL) defines the presence of Monoclonal B Lymphocytes in the blood of otherwise healthy individuals. Though phenotypically heterogeneous, most MBL cases resemble CLL cells (CD5+, CD20dim, CD79b dim, sIgdim). The interest in MBL increased after this entity was included in the revised NCI-WG/IWCLL guidelines for the diagnosis and management of CLL (Hallek et al, 2008) and defined as “the presence of fewer than 5×109/L B lymphocytes” in the peripheral blood. However, the concentration of MBL in the blood of any given individual is extremely variable accounting in some cases for the vast majority of circulating B cells while being a negligible portion of them in others. It is then plausible that molecular differences could exist between low- vs. high-count MBL, being the latter likely more advanced on the way to become CLL. In this context, it was recently reported that subjects with <5×109/L CLL-like MBL but with lymphocytosis will require treatment at a rate of 1.1% per year. The absolute B-cell count turned out to be the only independent prognostic factor associated with progressive lymphocytosis, as all MBL cases studied had immunoglobulin (IG) gene features and cytogenetic abnormalities similar to good prognosis CLL. In contrast, very little is known about low-count MBL cases accidentally found in the general population. By cytofluorograph analysis, we identified 89 CLL-like MBL in the blood of 1725 healthy individuals >18 years old (5.1%) and analyzed the IGHV-D-J rearrangements expressed by 51 of them, the majority being characterized by few clonal B cells (mean 6.9% of circulating B lymphocytes, with only 13 cases >10%). CLL-like MBL cells showed a predominance of IGHV3 genes, followed by IGHV4 genes, resembling the normal repertoire. The most frequent IGHV gene in MBL was IGHV4- 59/61, rarely used in CLL. The MBL repertoire was also conspicuous for the lack of the IGHV1-69 gene (the predominant gene in unmutated CLL, ~25%) and the low frequency of the IGHV4-34 gene (2/51 sequences, 3.9%), the most frequent gene in mutated CLL (~12%). Following the 98% identity cut-off value, 36/51 sequences (70.5%) were defined as “mutated”, whereas the remainder had “unmutated” IGHV genes. Alignment of MBL HCDR3 sequences to a comprehensive panel of CLL HCDR3 sequences identified 2/51 (3.9%) MBL cases with a sequence similar to previously described CLL cases (“stereotyped receptors”). These results show that the IG gene repertoire in low-count MBL, accidentally found in the general population, does not show the typical CLL-related biases in terms of IGHV gene usage. This cannot be simply explained by the higher number of mutated cases among MBL, as unmutated cases account for almost a third of CLL-like MBL, indicating a molecular heterogeneity. In addition, HCDR3 stereotypy in MBL is significantly less frequent than in CLL (>25% of cases). Occasional MBL may indeed express a CLL “stereotyped receptor”, implying that the potential to evolve into a leukemia exists within MBL, though at low frequency, and may depend on precise selection mechanisms based on the molecular features of the B cell receptor. Taken together, our results strongly suggest that the detection of MBL in an otherwise healthy subject is not always equivalent to a pre-leukemic state. Differential IG molecular features might provide a better tool to discriminate individuals at risk of progression than an arbitrary mathematical threshold. Detailed IG molecular analysis of individual MBL may help to identify those few cases that necessitate continuous clinical monitoring to anticipate disease progression and, on the other hand, to avoid the burden of lengthy and expensive follow-ups in the vast number of persons who are extremely unlikely to develop CLL, though carrying MBL in their blood.

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The immunoglobulin gene repertoire of low-count chronic lymphocytic leukemia (CLL)–like monoclonal B lymphocytosis is different from CLL: diagnostic implications for clinical monitoring

Blood ◽

10.1182/blood-2008-09-176933 ◽

2009 ◽

Vol 114 (1) ◽

pp. 26-32 ◽

Cited By ~ 96

Author(s):

Antonis Dagklis ◽

Claudia Fazi ◽

Cinzia Sala ◽

Valeria Cantarelli ◽

Cristina Scielzo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

International Workshop ◽

Good Prognosis ◽

Lymphocytic Leukemia ◽

Clinical Monitoring ◽

Gene Repertoire ◽

Immunoglobulin Gene ◽

Healthy Individuals ◽

Ighv Gene

Abstract In the revised National Cancer Institute Working Group (NCI-WG)/International Workshop on Chronic Lymphocytic Leukemia (IWCLL) guidelines for CLL, CLL-like monoclonal B lymphocytosis (MBL) is defined as the presence of less than 5 × 109/L B lymphocytes in the peripheral blood. However, the concentration of MBL in the blood is extremely variable. MBL in subjects with lymphocytosis require treatment at a rate of 1.1% per year and present immunoglobulin (IG) gene features and similar to good prognosis CLL. Little is known about low-count MBL cases, accidentally found in the general population. We analyzed IGHV-D-J rearrangements in 51 CLL-like MBL cases from healthy individuals, characterized by few clonal B cells. Seventy percent of the IGHV genes were mutated. The most frequent IGHV gene was IGHV4-59/61, rarely used in CLL, whereas the IGHV1–69 gene was lacking and the IGHV4-34 gene was infrequent. Only 2 of 51 (3.9%) MBL cases expressed a CLL-specific stereotyped HCDR3. Therefore, the IG gene repertoire in low-count MBL differs from both mutated and unmutated CLL, suggesting that the detection of MBL in an otherwise healthy subject is not always equivalent to a preleukemic state. Detailed IG analysis of individual MBL may help to identify cases that necessitate continuous clinical monitoring to anticipate disease progression.

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Immunoglobulin Gene Repertoire in Ocular Adnexa Lymphomas (OAL): Hints on the Nature of the Antigenic Stimulation

Blood ◽

10.1182/blood.v112.11.623.623 ◽

2008 ◽

Vol 112 (11) ◽

pp. 623-623

Author(s):

Antonis Dagklis ◽

Maurilio Ponzoni ◽

Andres JM Ferreri ◽

Maria Giulia Cangi ◽

Lorenza Pecciarini ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Lymphocytic Leukemia ◽

Thyroid Peroxidase ◽

Antigen Binding ◽

Gene Repertoire ◽

Immunoglobulin Gene ◽

Antigen Binding Site ◽

Significant Similarity ◽

Molecular Features

Abstract OALs arising in the ocular adnexae account for 15% of all extranodal lymphomas and are mostly represented by mucosa-associated lymphoid tissue (MALT)-type lymphomas (OAML). OAML have been described to be significantly associated, although with a variable geographic distribution, with Chlamydiophila psittaci (CP) infection, the etiologic agent of human psittacosis. Though the role played by Chlamydiae in OAML pathogenesis is also strongly supported by the observation of clinical remissions after bacterial eradication alone, the actual mechanisms have not been yet elucidated. Previous analysis of the Immunoglobulin Heavy Chain Variable (IGHV) genes in OAML showed a restricted repertoire with mutated genes and ongoing somatic mutations, thereby alluding to an origin from an antigen–experienced B cell, which has undergone antigen-selection; however, data on the potential antigenic elements stimulating OAML B lymphocytes are not available. In order to get hints on the potential ligands, we aimed at investigating the Immunoglobulin structure and, in particular, the molecular features of the antigen binding site (i.e. the HCDR3) of IG genes expressed by OAML. We analyzed the monoclonal IGHV-D-J rearrangements in a series of 30 OAML cases. IGHV3 subgroup genes predominated (24/30, 80%), followed by IGHV4 (4/30, 13.3%) and IGHV1 (2/30, 6,7%) subgroup genes, in keeping with the normal B cell IG gene repertoire. According to the 98% identity cut-off value, 23/30 sequences (76,7%) were defined as “mutated”, with a percentage of identity ranging between 89.3% and 97.7%, whereas the remainder (7/30 sequences; 23.3%) had “unmutated” IGHV genes. At individual gene level, IGHV3-23 and IGHV3-43 were the most frequently used genes (6/30 sequences, 20%, and 4/30 sequences, 13,3%, respectively); interestingly, the latter is rarely used in the normal repertoire. No significant correlations were found between IGHV gene usage and CP infection. HCDR3 length ranged from 9 to 30 amino acids (median 16) and 20/28 HCDR3 sequences showed an isoelectric point (pI) value >6.0, (in 10/28 pI>8.0), a feature shared with antibodies with anti–DNA reactivity. Shared epitope recognition was excluded by cluster analysis of HCDR3 sequences, also among OAML cases using the same IGHV genes. When we aligned HCDR3 sequences to a panel of 8214 unique IGHVD- J sequences from various types of normal, autoreactive or malignant B cell clones, we observed significant similarity between 3 OAML cases and 2 auto-reactive antibodies, present in rheumatoid arthritis and Sjoegren’s syndrome, respectively, and 1 antibody with anti-thyroid peroxidase (TPO) reactivity. The latter case showed a HCDR3 similarity also with the IG gene of a Chronic Lymphocytic Leukemia case, a disease characterized by frequent expression of auto-reactive Igs. Interestingly, the same search performed using anti-Chlamydia antibodies did not show any significant similarity. In conclusion, these results suggest that OAML express a distinctive IG gene repertoire and may originate from B cells selected for their capacity to bind to auto-antigens in a similar way to what reported for gastric MALT lymphomas, where malignant B cells, instead of directly reacting against Helicobacter Pylori, are actually stimulated by tissue auto-antigens exposed during the inflammatory reaction, as confirmed by molecular analyses of antigen binding sites of the monoclonal IG. Our findings support the hypothesis that stimulation by auto-antigens, likely generated within an inflammatory background promoted by Chlamydial infection, may be the driving force also in OAML pathogenesis, rather than a direct action of the bacteria on B cells.

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CLL-Like MBL In the General Population Persist Over Time, without Clinical Progression, Though Carrying the Same Cytogenetic Abnormalities of CLL

Blood ◽

10.1182/blood.v116.21.2440.2440 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2440-2440 ◽

Cited By ~ 1

Author(s):

Paolo Ghia ◽

Claudia Fazi ◽

Lorenza Pecciarini ◽

Lydia Scarfò ◽

Francesca Cottini ◽

...

Keyword(s):

B Cells ◽

General Population ◽

B Cell ◽

Potential Risk ◽

Healthy Individuals ◽

Follow Up Study ◽

Risk Of Progression ◽

Trisomy 12 ◽

Over Time

Abstract Abstract 2440 Monoclonal B-cell Lymphocytosis (MBL) is a preclinical hematologic condition wherein small B-cell clones are detectable in the peripheral blood of otherwise healthy individuals. Monoclonal B-cell expansions are rather heterogeneous in terms of phenotype, but in two thirds of cases clonal B-cell populations share the same unique immunophenotypic profile of Chronic Lymphocytic Leukemia (“CLL-like MBL”). Based on the number of B cells per μl, MBL cases might be further split into those associated with lymphocytosis, usually diagnosed in a clinical setting (“Clinical MBL”) when clonal B cells reach a concentration >1500/μL, and those detected in the general population (“low-count MBL”), usually characterised by <50 aberrant B cells per μl. It has been proposed that these two entities differ in terms of molecular and clinical features including prognosis. In the case of MBL with lymphocytosis, it has been previously shown and confirmed that they carry a potential risk of progression into clinically overt CLL of about 1.1% per year. On the contrary, very limited data about the outcome of MBL in the general population (“low-count”) exist, though these are the most common forms in otherwise healthy individuals (>20% of people older than 60 years using highly sensitive techniques). We took advantage of our cohort of 138 MBL cases previously described among 1779 healthy individuals, living in a rural valley in Northern Italy (Val Borbera Valley) that included 96 CLL-like (69.6%), 20 Non-CLL (14.5%), 22 atypical CLL (15.9%) MBL. Of the 138 originally diagnosed MBL subjects, 76 individuals participated to a second visit after a median follow up of 34 months (range 11–50 months). 93.1% (54/58) of CLL-like MBL clones were confirmed, while only 44.5% and 66.7% of Atypical CLL-like and Non-CLL MBL, respectively, persisted over time. The few CLL-like clones that were not confirmed had a very low concentration at the initial visit (median number of clonal B-cells: 0.46 per μl) being proximal to the detection limit of the flow cytometric technique. Among the confirmed CLL-like cases, 1/54 was a Clinical MBL (1764 cells/μL), 3 subjects had 97, 190 and 265 cells/μl, while the vast majority of participants (50/54, 92.6%), had a number of monoclonal B-cells <50/μl. In comparison with the initial evaluation, during the follow-up analysis, no CLL-like MBL developed a frank leukemia and, in particular, all B-cell expansions with <50 cells per μl remained stable or decreased in terms of absolute count, though some changes occurred in terms of percentage due to a decrease in the normal B cell population as an aging effect. The 3 clones with more than 50 cells per μl at the first evaluation, showed a variable increase in the number of aberrant cells per μL, though all remained below 400 cells/μL; the single clinical MBL case did not show any significant increase. In order to get further insights in the molecular features of the “low-count MBL”, FACS-sorted aberrant B-cells of 17 cases were subsequently studied by Fluorescence in situ Hybridization (FISH) for the most frequent genomic aberrations detected in CLL patients (del13q, trisomy 12, del11q, del17p). Interestingly, about half of the cases studied (8/17: 47.1%) showed mono- or bi-allelic (in 2 cases) 13q deletions, in a median of 26.7% of MBL cells. One of these cases carried also a 17p deletion that was detected in an additional individual; trisomy 12 and chromosomal deletion of 11q were not identified. In conclusion, our follow-up study in the general population show that CLL-like MBL tend to persist over time, in clear contrast with Non-CLL and Atypical CLL MBL that appear to be more transient, likely depending on a concomitant inflammatory/infectious status. Interestingly, though we previously reported that low-count CLL-like MBL express an Immunoglobulin repertoire different from clinic MBL and overt CLL, we here show that they do carry 13q deletions in frequency identical to CLL, suggesting the occurrence of this abnormality early during ontogenesis, likely associated with the acquisition of the typical phenotype rather than the progression into an overt leukemic disease. Finally, this follow-up study suggests that the potential risk of progression into clinically frank CLL for population-screening (“low-count”) CLL-like MBL is exceedingly rare if any and definitely less than that of individuals with clinical MBL. Disclosures: No relevant conflicts of interest to declare.

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Histopathologic and Molecular Features of Persistent Polyclonal B-Cell Lymphocytosis Challenging the Benign Nature of the Disorder.

Blood ◽

10.1182/blood.v108.11.4612.4612 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4612-4612

Author(s):

Ilaria Del Giudice ◽

Stefano A. Pileri ◽

Maura Rossi ◽

Elena Sabattini ◽

Cristina Campidelli ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Pcr Analysis ◽

White Pulp ◽

Splenic Marginal Zone Lymphoma ◽

Molecular Features ◽

Lymphoid Infiltration ◽

Igh Rearrangement

Abstract Persistent polyclonal B-cell lymphocytosis (PPBL) is an unusual expansion of B lymphocytes, whose immunophenotype resembles that of memory B cells. The physiopathology of PPBL is unknown and whether this syndrome represents a pre-malignant or benign disease is unclear. Aiming at further defining the histological features of BM involvement and at challenging the polyclonal nature of PPBL, we reviewed 6 cases of PPBL who underwent BM biopsy. There were 3 males and 3 females, with a median age at diagnosis of 35 years (range 17–46). The median lymphocyte count was 5.3 × 109/L (5.1–8.3). One patient had been splenectomized 10 years earlier and 2 were splenomegalic. During the follow-up (median 20 months, range 5–138), 1 showed progressive spleen enlargement and 2 additional patients developed splenomegaly. Screening for hepatitis B, C and HIV was negative. PB morphology showed 2 to 32% of cleaved lymphocytes. Immunophenotype documented 21 to 70% B lymphocytes in both PB and BM aspirate, all positive for CD19, CD22, CD79b, FMC7, CD18 and CD11a, negative for CD5, CD23, CD10, with no Ig light chain restriction. CD25 was expressed in 4 cases. PCR analysis of the IgH gene configuration of PB and BM cells showed a polyclonal picture in all cases. On Giemsa-stained sections of BM trephines, BM was normocellular in 5 cases and slightly hypocellular in 1. A mild lymphoid infiltrate was visible, mainly interstitial with occasional small lymphoid aggregates. An intrasinusoidal pattern was seen in 4/6 cases. Immunohistochemistry confirmed the interstitial infiltration by B cells (CD79a+, CD20+, IgM+, IgD+, DBA44−/+, CD5−, AnnexinD1−) in all cases and highlighted the intrasinusoidal pattern in 4 in which a splenic marginal zone lymphoma (MZL)-like morphology was described. By IgH gene PCR on BM tissue, a clonal rearrangement was detected in 2/4 cases with intrasinusoidal infiltration; in 2 cases DNA quality was poor. The 4 cases showed splenomegaly at diagnosis or during the follow-up. Splenectomy was performed in 1 patient due to progressive enlargement of the spleen over 9 years. White pulp was normal and red pulp was expanded by an infiltration of small lymphocytes with an intrasinusoidal distribution. Splenic B lymphocytes were CD79a+, CD20+, IgM+, IgD+, DBA44−, as in the BM biopsy, with low proliferation activity and proven clonality by IgH-PCR. In the remaining 2 cases, in which no intrasinusoidal lymphoid pattern was evident, clonal IgH rearrangement was absent and no splenomegaly has developed. We confirm the interstitial lymphoid infiltration of BM trephine in all cases of PPBL, with an evident intrasinusoidal component in some. The detection of IgH rearrangements on lymphocytes infiltrating the BM from bioptic samples and the absence of clonality in PB lymphocytes and in the BM aspirate, possibly due to the intrasinusoidal distribution, have not been described. These findings suggest that at least some of the cases described as PPBL are pre-malignant disorders close to splenic MZL, justifying the occurrence of progressive splenomegaly over time. Prolonged follow-up of patients with PPBL and an accurate biologic work-up of the BM, are therefore warranted.

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AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2482 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1046.1-1046

Author(s):

L. Schlicher ◽

P. Kulig ◽

M. Murphy ◽

M. Keller

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Cell Activation ◽

Surface Expression ◽

Cell Surface Expression ◽

B Cell Activation ◽

Human B Cells ◽

Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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Diffuse Large B-Cell Lymphoma: Recognition of Markers for Targeted Therapy

Hemato ◽

10.3390/hemato2020017 ◽

2021 ◽

Vol 2 (2) ◽

pp. 281-304

Author(s):

Laura Tomas-Roca ◽

Marta Rodriguez ◽

Ruth Alonso-Alonso ◽

Socorro M. Rodriguez-Pinilla ◽

Miguel Angel Piris

Keyword(s):

Targeted Therapy ◽

B Cells ◽

B Cell ◽

B Cell Lymphoma ◽

Therapeutic Approaches ◽

Molecular Features ◽

Molecular Variants ◽

The Status ◽

Underlying Causes ◽

Large B Cell

Diffuse large B-cell lymphomas (DLBCL)s, the most common type of Non-Hodgkin’s Lymphoma, constitute a heterogeneous group of disorders including different disease sites, strikingly diverse molecular features and a profound variability in the clinical behavior. Molecular studies and clinical trials have partially revealed the underlying causes for this variability and have made possible the recognition of some molecular variants susceptible of specific therapeutic approaches. The main histogenetic groups include the germinal center, activated B cells, thymic B cells and terminally differentiated B cells, a basic scheme where the large majority of DLBCL cases can be ascribed. The nodal/extranodal origin, specific mutational changes and microenvironment peculiarities provide additional layers of complexity. Here, we summarize the status of the knowledge and make some specific proposals for addressing the future development of targeted therapy for DLBC cases.

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Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

Blood ◽

10.1182/blood-2020-134572 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Misaki Sugai ◽

Naohiro Tsuyama ◽

Yu Abe ◽

Yusuke Azami ◽

Kenichi Kudo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

B Lymphocytes ◽

Research Funding ◽

Chromosomal Translocation ◽

Stem Cell Differentiation ◽

Advisory Committees ◽

Myeloma Cells ◽

Company Limited

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 & MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ & AX) and MIB2-6 t(11;14) (BC & BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Interleukin-13 in Combination With CD40 Ligand Potently Inhibits Apoptosis in Human B Lymphocytes: Upregulation of Bcl-xL and Mcl-1

Blood ◽

10.1182/blood.v89.12.4415 ◽

1997 ◽

Vol 89 (12) ◽

pp. 4415-4424 ◽

Cited By ~ 54

Author(s):

Jon Lømo ◽

Heidi Kiil Blomhoff ◽

Sten Eirik Jacobsen ◽

Stanislaw Krajewski ◽

John C. Reed ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Peripheral Blood ◽

Cell Apoptosis ◽

Cell Types ◽

Cd40 Ligand ◽

Interleukin 13

Abstract Interleukin-13 (IL-13) is a novel T-cell–derived cytokine with IL-4–like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

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B lymphocytes express and lose syndecan at specific stages of differentiation.

Cell Regulation ◽

10.1091/mbc.1.1.27 ◽

1989 ◽

Vol 1 (1) ◽

pp. 27-35 ◽

Cited By ~ 272

Author(s):

R D Sanderson ◽

P Lalor ◽

M Bernfield

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

B Lymphocyte ◽

Plasma Cells ◽

Receptor Expression ◽

Cell Stage ◽

Precursor B Cells

Lymphopoietic cells require interactions with bone marrow stroma for normal maturation and show changes in adhesion to matrix during their differentiation. Syndecan, a heparan sulfate-rich integral membrane proteoglycan, functions as a matrix receptor by binding cells to interstitial collagens, fibronectin, and thrombospondin. Therefore, we asked whether syndecan was present on the surface of lymphopoietic cells. In bone marrow, we find syndecan only on precursor B cells. Expression changes with pre-B cell maturation in the marrow and with B-lymphocyte differentiation to plasma cells in interstitial matrices. Syndecan on B cell precursors is more heterogeneous and slightly larger than on plasma cells. Syndecan 1) is lost immediately before maturation and release of B lymphocytes into the circulation, 2) is absent on circulating and peripheral B lymphocytes, and 3) is reexpressed upon their differentiation into immobilized plasma cells. Thus, syndecan is expressed only when and where B lymphocytes associate with extracellular matrix. These results indicate that B cells differentiating in vivo alter their matrix receptor expression and suggest a role for syndecan in B cell stage-specific adhesion.

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A reservoir of rituximab-resistant splenic memory B cells contributes to relapses after B-cell depletion therapy

10.1101/833343 ◽

2019 ◽

Cited By ~ 1

Author(s):

Etienne Crickx ◽

Pascal Chappert ◽

Sandra Weller ◽

Aurélien Sokal ◽

Imane Azzaoui ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Plasma Cells ◽

Complete Response ◽

Memory B Cells ◽

Chain Gene ◽

Gene Repertoire ◽

Heavy Chain Gene ◽

Immunoglobulin Heavy Chain Gene ◽

Pathogenic Antibodies

AbstractImmune thrombocytopenia (ITP) is an autoimmune disease mediated by pathogenic antibodies directed against platelet antigens, including GPIIbIIIa. Taking advantage of spleen samples obtained from ITP patients, we characterized by multiples approaches the onset of disease relapses occurring after an initial complete response to rituximab. Analysis of splenic B cell immunoglobulin heavy chain gene repertoire at bulk level and from single anti-GPIIbIIIa B cells revealed that germinal centers were fueled by B cells originating from the ongoing lymphopoiesis, but also by rituximab-resistant memory B cells, both giving rise to anti-GPIIbIIIa plasma cells. We identified a population of splenic memory B cells that resisted rituximab through acquisition of a unique phenotype and contributed to relapses, providing a new target in B cell mediated autoimmune diseases.

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