Normal B Cell-Derived iPSCs Capable of Inducing RAS Mutants and Aid to Explore Myeloma-Initiating Cells

Abstract Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and induce the expression of activation-induced cytidine deaminase (AID) by the Tet-off system (Sci Rep, 2017). Subsequently, we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5′-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14) carrying the DJ-rearranged allele of IgH that reciprocally translocated with CCND1 (G28). All these BiPSCs differentiated into hematopoietic progenitor cells (HPCs) (Sci Res, 2021). Next, we created AID fused with the hormone-binding domain of the estrogen receptor T2 mutant (AID-ER) to analyze the function of AID. The ER sequence of pCAG-ERT2CreERT2 (Addgene # 13777) was amplified by PCR and cloned into the downstream part of the TREtight promoter of pRetroX-Tight-Hyg as an open reading frame using an In-Fusion HD cloning kit. The ER sequence was also cloned into the same vector as a negative control for the analysis of AID function. These vectors were packaged into a retrovirus using gp293 and pMD2.G, and the viruses were infected into HEK293 cells expressing Tet-off transcription factor and fluorescence-deficient EGFP, in which the artificially-introduced stop codon was at the 106 thcodon of the EGFP gene. The expression of AID-ER fusion protein was induced by removal of doxycycline three days before analysis. AID activity was induced by the addition of 4-hydroxytamoxifen, an agonist of ER mutants, one day before analysis. Therefore, AID activity was monitored by detecting the frequency of EGFP-positive cells, i.e., the recovery of fluorescence, by flow-cytometry. Mutation of the artificial stop codon (from TAG to TAC; from stop codon to Tyrosine) was confirmed by sequencing the EGFP DNA amplified by PCR from the sorted EGFP-positive cells. Subsequently, we examined the introduction of mutated KRAS and NRAS genes into G28, because the most frequently occurring mutations in patients with MM are in KRAS and NRAS. Mutated KRAS (G12A) and NRAS (G13D) were amplified by RT-PCR from poly (A) mRNA of MM.1S and KMM1 of MM cell lines, respectively. After confirming the sequence of these mutants, the mutant cDNA of KRAS or NRAS was cloned into pRetroX-Tight-Hyg in combination with AID-ER. These two vectors were packaged into retroviruses, following which the viruses were infected into BiPSC13 with t(11;14) expressing Tet-off transcription factors, respectively. These BiPSCs also differentiated into HPCs. However, unlike cord blood, these HPCs did not differentiate into B lymphocytes by co-culture with BM stromal cell (MS-5). Therefore, further ingenuity is required to differentiate the BiPSC-derived HPCs into B lymphocytes. Disclosures No relevant conflicts of interest to declare.

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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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Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells

Scientific Reports ◽

10.1038/s41598-021-84628-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yusuke Azami ◽

Naohiro Tsuyama ◽

Yu Abe ◽

Misaki Sugai-Takahashi ◽

Ken-ichi Kudo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Pluripotent Stem Cells ◽

Stromal Cell ◽

Chromosomal Translocation ◽

Ips Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Induced Pluripotent

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Immunological Shift On Day 15 of Remission Induction in Children with CD10-Positive B-Cell Precursor ALL Treated by ALL-MB 2008 Protocol.

Blood ◽

10.1182/blood.v114.22.2650.2650 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2650-2650

Author(s):

Alexander Popov ◽

Tatiana Verzhbitskaya ◽

Grigory Tsaur ◽

Egor Shorikov ◽

Leonid Saveliev ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Fluorescence Intensity ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Remission Induction ◽

Cell Precursor ◽

Childhood All

Abstract Abstract 2650 Poster Board II-626 Statement. Flow cytometric (FC) minimal residual disease (MRD) monitoring during remission induction is a strong tool for prediction of outcome in children with acute lymphoblastic leukemia (ALL) of B-cell precursor (BCP) origin. Antigens' expression changes during treatment. Immunological shift can significantly complicate MRD detection. Immunophenotypical changes could vary during antileukemic treatment. Aim. To compare CD19, CD10, CD34, CD20, CD45 and CD58 expression at diagnosis and on day 15 of remission induction of currently applied ALL-MB 2008 treatment protocol and to evaluate significance of this changes for FC MRD monitoring. Patients and methods. Since November 2008 till July 2009 38 cases of childhood ALL were enrolled onto ALL-MB 2008 trial in Pediatric Oncology and Hematology Center in Ekaterinburg. Among them 22 patients were selected for present study as they fulfilled following criteria: had CD10-positive BCP-ALL and were MRD-positive ≥ 10−4 on day 15. MRD detection in bone marrow was performed by 9-color FC. We compared mean fluorescence intensity (MFI) values at diagnosis and on day 15. We also analyzed changes in CD19, CD20, CD45 and CD58 expression by normal B-lymphocytes on day 15 because at this time-point B-lineage regeneration is absent in bone marrow and MRD has to be discriminated only from mature B-cells. Coefficient of variation (CV) of fluorescence intensity and percentage of positive cells were used for evaluation of cells' distribution changes. Results. At diagnosis we found significant differences in antigen expression between tumor cells and B-lymphocytes. CD10, CD34, CD58 overexpression and CD19, CD20, CD45 underexpression by leukemic blasts were noted. On day 15 CD10, CD34 and CD58 were downmodulated (p = 0,0030, p = 0,0007 and p = 0,0017 respectively) while CD19, CD20 and CD45 were upmodulated (p = 0,0033, p = 0,0273 and p = 0,0001 respectively). At the same time CD20, CD45 and CD58 expression by mature B-lymphocytes decreased (p = 0,0013, p = 0,0130 and p = 0,0067 respectively) while CD19 was stable (p = 0,1060). Hence malignant and normal cells became closer on dot plots. On day 15 significant differences in CD10, CD34, CD20, CD45 and CD58 between blasts and lymphocytes were still presented. CD19 expression by leukemic cells on day 15 became higher than by mature B-cells (median MFI = 20284, range1587-34505 and median MFI = 15823, range 811–30882, p = 0,0196). Cells' distribution by CD10 and CD34 became more heterogeneous (p = 0,0364 and p = 0,0008 respectively), CD45 expression became more homogeneous (p = 0,0003) while CD20 and CD58 expression CV didn't change significantly (p = 0,1913 and p = 0,3443 respectively). CD20- and CD45-positive cells' percentage increased (p = 0,0015 and p = 0,0002 respectively) and CD34-positive cells' percentages decreased (p = 0,0003). Discussion. Changes in antigens' expression could complicate MRD detection so far as they decrease immunophenotypical differences between tumor blasts and mature B-lymphocytes. Interestingly, disparity of our results and previously shown data was found. Differences in immunological shift could be partially explained by differences in induction intensity of ALL-MB 2008 and international protocols. Conclusion. Significant immunological shift occurred on day 15 of ALL-MB 2008 remission induction. These antigens' expression changes have to be taken into account by the researcher for the successful FC MRD monitoring. Disclosures: No relevant conflicts of interest to declare.

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ZAP-70 Enhances BCR Signaling and Migration of Malignant B Lymphocytes towards CCL21 Via Induction of CCR7 Expression

Blood ◽

10.1182/blood.v116.21.3591.3591 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3591-3591

Author(s):

Eva Calpe ◽

Maria Joao Baptista ◽

Carles Codony ◽

Neus Villamor ◽

Pau Abrisqueta ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Cell Lines ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Prognostic Features ◽

Ccr7 Expression ◽

Bcr Signaling ◽

And Migration

Abstract Abstract 3591 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) and the effects in BCR signaling and migration were studied. BCR-expressing B-cells (Ramos) where stimulated with F(ab’)2 anti-IgM or F(ab’)2 anti-IgD. IgM but not IgD stimulation induced ZAP-70 activation and mobilization to the membrane, which, in turn, enhanced signaling through phosphorilation of AKT and ERK1-2 kinases. The presence of ZAP-70 also inhibited IgM and CD79b internalization after BCR stimulation, thus allowing for a longer and sustained stimulation. In Raji B-cells, which did not express BCR, ZAP-70 was constitutively phosphorilated, which induced a higher level of ERK1-2 phosphorilation only, suggesting that AKT phosphorilation in Ramos expressing ZAP-70 after IgM stimulation was depending on additional factors. BCR stimulation in Ramos B cells changed the expression pattern of several cytokine receptors such as CXCR4 and CXCR3 (downregulated) and CCR7 (upregulated). Interestingly, ZAP-70 activation was involved in the induction of CCR7 expression, which was significantly upregulated upon BCR stimulation in ZAP-70 expressing B-cells. This upregulation was also confirmed in Raji B cells upon ZAP-70 expression and activation. The relationship between ZAP-70 and CCR7 was also seen in neoplastic B-lymphocytes from patients with CLL where there was a linear correlation between the expressions of the two molecules. The increased CCR7 expression in ZAP-70 expressing B-cell lines translated into an enhanced signaling though CCR7 upon CCL21 addition and an enhanced migration towards a CCL21-containing medium in vitro. In conclusion, ZAP-70 ectopic expression leaded to an enhanced signaling through the BCR after IgM stimulation while it did not participate in the signaling through IgD. Moreover, ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21. These results give further insight into the functional role of ZAP-70 protein in the aggressiveness of CLL cases with an increased expression of ZAP-70. Disclosures: No relevant conflicts of interest to declare.

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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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P54 Pneumocystis jirovecii prophylaxis in patients on rituximab

Rheumatology ◽

10.1093/rheumatology/kez416.021 ◽

2019 ◽

Vol 58 (Supplement_4) ◽

Cited By ~ 1

Author(s):

Kishore Warrier1 ◽

Catherine Salvesani ◽

Samundeeswari Deepak

Keyword(s):

Risk Factors ◽

T Cell ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Pneumocystis Jirovecii ◽

Current Evidence ◽

Pneumocystis Jirovecii Pneumonia ◽

Number Of Patients ◽

Increased Risk

Abstract Background Rituximab is a chimeric monoclonal antibody that depletes the B cell population by targeting cells bearing the CD20 surface marker and is used widely in the management of paediatric rheumatological conditions like juvenile systemic lupus erythematosus (JSLE), juvenile dermatomyositis (JDM), mixed connective tissue disease (MCTD) and juvenile idiopathic arthritis (JIA). Pneumocystis jirovecii pneumonia (PCP) is a potentially fatal opportunistic infection associated with congenital and acquired defects in T cell–mediated immunity. Our guideline did not recommend prophylaxis against PCP for patients on rituximab, unlike patients on cyclophosphamide, who are on cotrimoxazole until three months after cessation of the treatment. Cyclophosphamide is an alkylating agent which affects both B and T lymphocytes. Following the death of 16 year-old girl with JSLE due to PCP, the team reviewed the possible contributing factors, undertook a review of literature and discussed this at multi-disciplinary meetings involving the microbiology and immunology teams. This patient was found to have other risk factors for PCP – low CD4 T cells, concomitant use of corticosteroids and hypogammaglobulinaemia (IgG 3.0g/L). Although there is limited evidence that rituximab on its own increases the risk of PCP, there is emerging data that B cells may have a role in the protection against pneumocystis. Following the review, it was concluded that children on rituximab and an additional immunosuppressant (including corticosteroids) should receive prophylactic cotrimoxazole to cover PCP. Methods Retrospective audit carried out by the team to look at adherence to the new guideline regarding the use of cotrimoxazole for PCP prophylaxis in patients who have had rituximab between August 2017 and May 2019. Results P54 Table 1 Total number of patients who had rituximab 10 Number of patients who had other immunosuppressants concomitantly / recently (within previous 3 months) 7 Number of patients on rituximab monotherapy 2 Number of patients who are 6 months post-treatment 1 Number of patients with other risk factors for PCP 1 (hypogammaglobulinaemia) Number of patients who are eligible for prophylaxis, as per the guideline 8 (7 for concomitant immunosuppression and 1 for hypogammaglobulinaemia) Number of patients on cotrimoxazole 7 (87.5%) - one of the patients is on methotrexate, which is advised not to combine with cotrimoxazole We achieved 87.5% compliance in prescribing cotrimoxazole for PCP prophylaxis to all rheumatology patients receiving rituximab alongside another immunosuppressant agent; the one patient who this was not adhered to was due to potential adverse drug pharmacodynamic interaction between cotrimoxazole and methotrexate. Conclusion Although the current evidence points to increased risk of PCP in patients with inherited and iatrogenic defect of T cell function, there is emerging evidence that B cells may have a role too. Hence more work is required to determine the risk of PCP in patients on B cell targeted therapy (BCTT) and the need for prophylaxis. Conflicts of Interest The authors declare no conflicts of interest.

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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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The RNase III enzyme Dicer is essential for germinal center B-cell formation

Blood ◽

10.1182/blood-2011-05-355412 ◽

2012 ◽

Vol 119 (3) ◽

pp. 767-776 ◽

Cited By ~ 64

Author(s):

Shengli Xu ◽

Ke Guo ◽

Qi Zeng ◽

Jianxin Huo ◽

Kong-Peng Lam

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Plasma Cells ◽

Cytidine Deaminase ◽

Cell Formation ◽

Terminal Differentiation ◽

Mature Mirnas ◽

Rnase Iii ◽

Proapoptotic Protein

Abstract MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are important for pre-B and follicular B lymphopoiesis as demonstrated, respectively, by mb-1-Cre– and cd19-Cre–mediated deletion of Dicer, the RNase III enzyme critical for generating mature miRNAs. To explore the role of miRNAs in B-cell terminal differentiation, we use Aicda-Cre to specifically delete Dicer in activated B cells where activation-induced cytidine deaminase is highly expressed. We demonstrate that mutant mice fail to produce high-affinity class-switched antibodies and generate memory B and long-lived plasma cells on immunization with a T cell–dependent antigen. More importantly, germinal center (GC) B-cell formation is drastically compromised in the absence of Dicer, as a result of defects in cell proliferation and survival. Dicer-deficient GC B cells express higher levels of cell cycle inhibitor genes and proapoptotic protein Bim. Ablation of Bim could partially rescue the defect in GC B-cell formation in Dicer-deficient mice. Taken together, our data suggest that Dicer and probably miRNAs are critical for GC B-cell formation during B-cell terminal differentiation.

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Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.

Journal of Experimental Medicine ◽

10.1084/jem.158.3.920 ◽

1983 ◽

Vol 158 (3) ◽

pp. 920-931 ◽

Cited By ~ 38

Author(s):

M H Nahm ◽

J W Paslay ◽

J M Davie

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

X Chromosome ◽

Cell Subset ◽

Phosphoglycerate Kinase ◽

Mutant Gene ◽

Abnormal Development ◽

Female Mice ◽

Chromosomal Genes

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.

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