scholarly journals CD19+ B Lymphocytes Are Predictive for Anti-Sars-Cov-2 Vaccination Response in Multiple Myeloma Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3788-3788
Author(s):  
Susanne Ghandili ◽  
Martin Schönlein ◽  
Heiko Becher ◽  
Christian Wiessner ◽  
Marc Lütgehetmann ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Research Funding ◽  
B Lymphocyte ◽  
Medicine Research ◽  
Cell Numbers ◽  
Vaccination Response ◽  
Antibody Titers

Abstract Introduction: Up to now,reliable results regarding the efficacy of anti-SARS-CoV-2 vaccines in patients with multiple myeloma (MM), especially under current myeloma-directed therapy, are scarcely available. Here, we report an analysis describing the level of post-vaccination antibody titers after the 1 stand 2 ndanti-SARS-CoV-2 vaccination depending on therapy, remission status, and B- and T-cell numbers in patients with MM and related plasma cell neoplasia. Methods: This observational single-center study included patients aged ≥18 years with diagnoses of MM, monoclonal gammopathies of clinical significance (MGCS), or systemic light-chain amyloidosis (AL) who were eligible for Anti-SARS-CoV-2 vaccination according to the International Myeloma Society recommendations. Patients with prior COVID-19 infections were excluded. Samples were analyzed for the presence of SARS-CoV-2 specific antibodies using the quantitative anti-spike IgG (SARS-CoV-2 spike RBD IgG, cut off ≥ 0.8 BAU/ml) according to manufacturer's recommendations. SARS-CoV-2 spike protein antibody titer (SP-AbT) were evaluated after at least 7 days after the 1 stand 2 ndvaccination, respectively. This study was performed between January 1 - July 15, 2021, at the University Medical Center Hamburg-Eppendorf, Germany, as part of the COVIDOUT trial (NCT04779346). All patients provided written informed consent. Aims of this study were to evaluate a possible correlation between SP-AbT and CD19+ B lymphocyte count, as well as to identify other factors impacting vaccination response. Results: 82 patients who received SARS-CoV-2 vaccines (including 67 patients with mRNA-, 8 with vector-based vaccines and 4 heterologous vaccinations) were included. 74 patients had diagnosis of MM, 4 of MGCS/smoldering MM and 4 of AL. Median age was 68 years (range 35-85) and 49 patients were male. In total, 37 patients (45.1%) received anti-CD38- and 2 (2.4%) anti-SLAMF7-targeting therapies at the time of vaccination, 52 (63.4%) patients received immunomodulatory drug (IMID)-based treatments and 13 patients (15.9%) were under active surveillance. 59% of patients had newly diagnosed and 41% refractory or relapsed disease. In total, 75.6% of all patients were in deep remissions (very good partial remission or better). Assessment of anti-SARS-CoV-2 antibody titers took place in median 23 days (range [r] 8-63 days) after the 1 stand 21 days (r: 6-53) after the 2 ndvaccination. A positive SARS-CoV-2 SP-AbT was detected in 31.9% of assessable patients with an overall median SP-AbT of 0 BAU/ml (r: 0-10328, mean 202.36) after the 1 stvaccination and increased up to 88.9% (median SP-AbT of 216.87 BAU/ml, r: 0-25720, mean 2139.29) after 2 ndvaccination. Of the patients not showing positive SP-AbT after the 1 stvaccination, 80.9% became positive after 2 ndvaccination, while 19.1 % remained negative. Median SP-AbT titer was significantly lower compared to patients who became positive already after 1 stvaccination (51.04 vs. 2191.87 BAU/ml, p<0.0001). Regarding immune status, a CD19+ B cell count of median 33.5/µl (r: 1-696/µl) was seen in the overall patient cohort; in patients with negative SP-AbT, median CD19+ B cell numbers were significantly lower compared to patients with positive titers (median CD19+ B cells: 2.0 vs. 52.5/µl, p=0.005). Overall, CD19+ B lymphocyte numbers correlate significantly with positive SP-AbT results and were identified as predictive factor in multivariate analysis. The previously suggested threshold of 30 CD19+ B cells/µl as being predictive for SP-AbT development could be validated. SP-AbT concentration was significantly lower with older age. Furthermore, median SP-AbT were significantly lower in patients with current anti-CD38 directed therapy (median SP-AbT: 1085.4 vs. 62.05 BAU/ml, p < 0.005). Conclusions: In spite of immunodeficiency and immunosuppressive therapy, most MM patients develop SP-AbT. However, about 11% of MM patients failed to develop SP-AbT after full vaccination, and thus remain on risk for COVID-19. Higher counts of CD19+ B lymphocytes, with a threshold of 30 CD19+ B lymphocytes/µl, are predictive for SP-AbT formation and may further help to identify patients at higher risk of insufficient vaccination response in whom control of vaccination success and potential third vaccination are particularly important. Disclosures Bokemeyer: GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Eisai: Research Funding; Daiichi Sankyo: Research Funding; Gilead Sciences: Research Funding; Blueprint Medicine: Research Funding; BerGenBio: Research Funding; Janssen-Cilag: Research Funding; Isofol Medical: Research Funding; AOK Health insurance: Consultancy; GSO: Consultancy; Bayer Schering Pharma: Consultancy; Gylcotope GmbH: Research Funding; ADC Therapeutics: Research Funding; Apellis Pharmaceuticals: Research Funding; Amgen: Research Funding; Alexion Pharmaceuticals: Research Funding; Agile Therapeutics: Research Funding; Merck Serono: Consultancy, Other: Travel accomodation ; Lilly/ImClone: Consultancy; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; BMS: Honoraria, Other: Travel accomodation, Research Funding; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck KGaA: Honoraria; Abbvie: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Astellas: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Sinn: Incyte: Honoraria, Research Funding; Pfizer: Honoraria; Servier: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Astra Zenica: Consultancy, Research Funding; MSD: Consultancy, Research Funding; Sanofi: Consultancy; Bayer: Research Funding; BMS: Honoraria, Research Funding. Leypoldt: GSK: Consultancy, Other: Meeting attendance ; Sanofi: Consultancy; Abbvie: Other: Meeting attendance . Weisel: Adaptiv Biotec: Consultancy; Abbvie: Consultancy; BMS: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Honoraria; Novartis: Honoraria; Oncopeptides: Consultancy, Honoraria; Pfizer: Honoraria; Roche: Honoraria; Takeda: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding.

scholarly journals 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 ◽  
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.

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

1989 ◽  
Vol 1 (1) ◽  
pp. 27-35 ◽  
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.

scholarly journals B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

1998 ◽  
Vol 187 (5) ◽  
pp. 753-762 ◽  
Author(s):  
Conrad C. Bleul ◽  
Joachim L. Schultze ◽  
Timothy A. Springer
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cxc Chemokine

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.

scholarly journals B Cells in Primary Membranous Nephropathy: Escape from Immune Tolerance and Implications for Patient Management

2021 ◽  
Vol 22 (24) ◽  
pp. 13560
Author(s):  
Benjamin Y. F. So ◽  
Desmond Y. H. Yap ◽  
Tak Mao Chan
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Membranous Nephropathy ◽  
Patient Management ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Peripheral Tolerance ◽  
Cell Tolerance ◽  
B Cell Tolerance

Membranous nephropathy (MN) is an important cause of nephrotic syndrome and chronic kidney disease (CKD) in adults. The pathogenic significance of B cells in MN is increasingly recognized, especially following the discovery of various autoantibodies that target specific podocytic antigens and the promising treatment responses seen with B cell depleting therapies. The presence of autoreactive B cells and autoantibodies that bind to antigens on podocyte surfaces are characteristic features of MN, and are the result of breaches in central and peripheral tolerance of B lymphocytes. These perturbations in B cell tolerance include altered B lymphocyte subsets, dysregulation of genes that govern immunoglobulin production, aberrant somatic hypermutation and co-stimulatory signalling, abnormal expression of B cell-related cytokines, and increased B cell infiltrates and organized tertiary lymphoid structures within the kidneys. An understanding of the role of B cell tolerance and homeostasis may have important implications for patient management in MN, as conventional immunosuppressive treatments and novel B cell-targeted therapies show distinct effects on proliferation, differentiation and reconstitution in different B cell subsets. Circulating B lymphocytes and related cytokines may serve as potential biomarkers for treatment selection, monitoring of therapeutic response and prediction of disease relapse. These recent advances in the understanding of B cell tolerance in MN have provided greater insight into its immunopathogenesis and potential novel strategies for disease monitoring and treatment.

IGH Repertoire Analysis In Multiple Myeloma (MM): Lack of Intra-Disease Homology and Occasional Clustering with Sequences of Other B-Cell Neoplasms Sharing Identical Geographical Origin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2951-2951
Author(s):  
Simone Ferrero ◽  
Daniela Capello ◽  
Mirija Svaldi ◽  
Daniela Drandi ◽  
Michela Boi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Somatic Hypermutation ◽  
Cell Repertoire ◽  
Non Hodgkin Lymphoma ◽  
B Cell Subsets

Abstract Abstract 2951 Background: The identification of stereotyped immunoglobulin (IG) receptors has improved our knowledge on the pathogenesis of several B-cell malignancies, suggesting the role of antigen-driven stimulation in chronic lymphocitic leukemia (CLL), marginal-zone lymphoma (MZL) and mantle-cell lymphoma (MCL). Multiple myeloma (MM) is a terminally-differentiated neoplasm no longer expressing surface IG; however some reports suggest the existence of early B-lymphocyte precursors which could be susceptible to antigen-driven stimulation. IG heavy chain (IGH) repertoire has not been extensively investigated in MM, with the largest available reports containing less than 80 complete sequences. Aims: To address this issue we created a database of MM IGH sequences including our institutional records (mostly derived from minimal residual disease studies) and sequences available from the literature. We planned a two-step analysis: a) first we characterized the MM repertoire and performed intra-MM clustering analysis; b) then we compared our MM series to a large public database of IGH sequences from neoplastic and non-neoplastic B-cells in search of similarities between MM sequences and other normal or neoplastic IGH repertoires. Patients and methods: 131 MM IGH genes were amplified and sequenced at our Institutions and belonged to Italian patients, while 214 MM IGH sequences from non-Italian patients were derived from published databases (NCBI-EMBL-IMGT/LIGM-DB) for a total of 345 fully interpretable MM sequences (out of 396). 28590 IGH sequences from other malignant and non-malignant B-cells were retrieved from the same public databases, including approximately 4500 CLL/Non-Hodgkin lymphoma (NHL) sequences and comprising 500 sequences from Italian patients. All sequences were analyzed using the IMGT database and tools (Lefranc et al., Nucleic Acid Res. 2005; http://imgt.cines.fr/) to identify IGHV-D-J gene usage, to assess the somatic hypermutation (SHM) rate and to identify HCDR3. HCDR3 aminoacidic sequences were aligned together using the ClustalX 2.0 software (Larkin et al., Bioinformatics, 2007; http://www.clustal.org/). Subsets of stereotyped IGH receptors were defined according to Stamatopoulos et al. (Blood, 2007). Result: IGHV analysis in MM was almost in keeping with the normal B-cell repertoire, showing a less remarkably biased IGH usage compared to CLL, MCL and MZL (with seven genes accounting for 40% of cases, compared to respectively five, three and two genes). However, a modest but significant over-representation of IGHV1-69, 2–5, 2–70, 3–21, 3–30-3, 3–43, 5–51 and 6-1 genes and under-representation of the IGHV1-18, 1–8, 3–30, 3–53 and 4–34 was noticed. The rate of somatic hypermutation in MM followed a Gaussian distribution with a median value of 7.8%. Intra-MM search for HCDR3 similarities never met minimal requirements for stereotyped receptors. When MM sequences were compared to non-MM database, only a minority of MM sequences (2.6%, n=9) clustered with sequences from lymphoid tumors and normal B-cells (figure 1A). In particular two non-Italian MM sequences clustered with previously characterized, uncommon CLL subsets (n.37 and n.71 according to Murray et al., Blood 2008). Moreover, novel provisional clusters were observed including three MM-CLL subsets, one MM-NHL subset, and three MM-normal B-cell subsets. While the MM-normal B-cell clusters involved non-Italian patients, we unexpectedly noticed that the four MM-CLL/MM-NHL clusters were composed exclusively of Italian patients, as shown in figure 1B, although Italian subjects represented less than 12% of the entire CLL-NHL database. Conclusion: The analysis of the largest currently available database of MM IGH sequences indicates the following: 1) MM IGH repertoire is closer to physiological distribution than that of CLL, MCL and MZL; 2) MM specific clusters do not occur to a frequency detectable with currently available databases; 3) 98% of MM sequences are not related to other “highly-clustered” lymphoproliferative disorders; 4) Uncommon clustering phenomena may follow a geographical rather than a disease-related pattern. Disclosures: No relevant conflicts of interest to declare.

Characterization of Clonotypic B Cell Progenitors in Bone Marrow of Multiple Myeloma Patients,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4002-4002
Author(s):  
Kelly Boucher ◽  
Nancy Parquet ◽  
Kenneth H. Shain ◽  
Rachid Baz ◽  
Melissa Alsina ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Research Funding ◽  
Cell Activity ◽  
Stem Cell Activity ◽  
B Cell Precursors

Abstract Abstract 4002 The lack of specific molecules to define malignant B progenitor cells in multiple myeloma (MM) has hampered the evaluation of minimal residual disease (MRD). We have identified a bone marrow (BM) CD138- subset that co-express CD19+ with identical κ or λ light chain (LC) restriction as the abnormal plasma cell (PC), as previously shown by others. The majority of LC restricted (LCR) B progenitors are CD19+/CD34- and <0.5% of whole BM (WBM) cells exhibit an immature phenotype: CD19+/CD34+ with aberrant CD27 expression. Immature B cell precursors are undetectable in peripheral blood (PB). LCR CD138-/CD19+ cells represent only 0.72± 0.5% of WBM in newly diagnosed patients (n=23) and do not increase (0.47± 0.51%) in patients with relapsed disease (n=21). The κ/λ LC ratio is 1.46±0.6 regardless of disease stage suggesting that conventional LC ratios for PCs (> 4 or <0.5) may not apply in B progenitors. LCR B progenitors (CD19+/34+ or CD19+/34-) are CD117+, Notch+ and Survivin+ as MM patient's hematopoietic stem cells (HSC). ALDH enzymatic activity is 3.1% (0.1-–7.26%) in LCR B cells. Flow sorted CD138+ did not grow in a colony formation assay (methylcellulose with PHA-LCM), whereas CD19+/CD34- or CD19+/CD34+ grew colonies with efficiency of 1:25,000 or 1:10000 respectively. Cells harvested from colonies have a lympho-plasmacytoid appearance and LCR B progenitors differentiated into CD138+ PC (80±5%) compared to HSC (10±5%). Colony efficiency was optimized (3 fold) using conditioned medium (CM) from HS5-stroma. Isolated CD138-/CD19+ cells were relatively bortezomib and melphalan resistant compared to CD138+ PC. We hypothesize that CD138-/CD19+/CD34+ cells contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of immature B cell precursors. Research studies of MM putative progenitor cells will allow developing novel treatments to eradicate potential MM MRD reservoir. Disclosures: Baz: Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding.

scholarly journals Negative selection of human germinal center B cells by prolonged BCR cross-linking.

1996 ◽  
Vol 183 (5) ◽  
pp. 2075-2085 ◽  
Author(s):  
L Galibert ◽  
N Burdin ◽  
C Barthélémy ◽  
G Meffre ◽  
I Durand ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Germinal Center ◽  
B Lymphocyte ◽  
Calcium Flux ◽  
Cross Linking ◽  
B Cell Differentiation ◽  
Selection Of

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.

scholarly journals Onset of Regulatory B Cells Occurs at Initial Stage of B Cell Dysfunction in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1780-1780 ◽  
Author(s):  
Zhongqing Zou ◽  
Tingting Guo ◽  
Jian Cui ◽  
Li Zhang ◽  
Ling Pan
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Natural Science ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
T Test ◽  
High Dose ◽  
Regulatory B Cells

Introduction :Multiple myeloma(MM) is caused by aggregation of clonal plasma cells, clinically presenting as evolvement in order from monoclonal gamma globulin disease (MGUS), smoldering MM (SMM), symptomatic MM to plasma cell leukemia. Regulatory B cells (Bregs), only a small immunosuppressive subgroup of B cells, have been recently identified in the setting of autoimmune diseases, immune thrombocytopenia (ITP) and gastric cancer . The role of Bregs in MM remains poorly defined. Here, the study was carried out on how Bregs correlate with evolution of MM, as well as how Bregs would be influenced by bortezomib, which is currently the first-line anti-MM agent. Methods :All patients met the International Myeloma Working Group (IMWG) Criteria for the Diagnosis of MM. Mononuclear cells (MNCs) were isolated from bone marrow(BM)but not peripheral blood(PB)at specified time points. Cell numbers were quantified by hemocytometer. The ratios of Bregs and B cells were delected by flow cytometry (FCM). Propidium iodide was widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic. The results were expressed as the mean ± SD. Comparisons between 2 groups were performed with Student's t-test. Multiple groups (≥3) were analyzed by one-way ANOVA, and paired groups were analyzed by two-way ANOVA or Student t test. Data were graphed and analyzed using GraphPad Prism 6.0. P &lt; 0.05 was considered statistically significant. Results: The study firstly found that Bregs' ratio increased at very beginning stage of MM and headed for extinction during progression of MM. It showed that Bregs' ratios were 11.7 ± 6.3%, 11 ± 8.6%, 15.8 ± 6.8%, and 4.9 ± 2.1% at stage of MGUS (n=5), SMM (n=4), newly diagnosed MM (NDMM) (n=9), and relapsed or refractory MM (RRMM) (n=6), respectively (p&lt;0.05). We then obtained mononuclear cells from NDMM samples. It revealed a positive correlation on both ratios and absolute numbers between Breg subgroup and B cell groups when B cells' ratio was higher than 5% of BMMNCs, whereas Bregs' ratio was rarely detected when B cells' ratio was lower than 5%. Subsequently, a retest was made to observe how Breg subgroup would be influenced by using bortezomib to target B-cell reservoir in MM. We added bortezomib of different concentrations to MNCs from NDMM samples within culture medium RPMI1640 (Ruikos Biotechnologies) for 24 hours (n=6). Bregs and B cells were totally killed when treated with high-dose bortezomib (n=3), while partially killed with low-dose bortezomib (n=3). To further explore how bortezomib could influence Bregs, another 6 samples were enrolled to demonstrate that the apoptotic absolute number of Bregs significantly increased after treatment of high-dose bortezomib (135615 ± 92085 v.s. 81132 ± 52908, P ≤ 0.05). Conclusions : Bregs were strongly entwined with the whole group of preserved B cell in MM. Bregs began to increase at very beginning stage of MM when B cells were preserved, accompanying with transition from MGUS to diagnostic MM. Bregs would substantially decreased while B-cell reservoir diminished during MM progression or by B cell targeted bortezomib. Disclosures Zou: the National Natural Science Foundation of China: Research Funding. Guo:the National Natural Science Foundation of China: Research Funding. Cui:the National Natural Science Foundation of China: Research Funding. Zhang:the National Natural Science Foundation of China: Research Funding. Pan:the National Natural Science Foundation of China: Research Funding.

scholarly journals B Cells of the Proliferative Fraction of CLL Clones Exhibit Activated B-Cell and Myeloid-Cell Signatures Suggesting Enhanced Antigen-Presentation, Integrin Responsiveness, and IL-4 Receptiveness: Additional Targets for CLL Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 302-302
Author(s):  
Xiao-Jie Yan ◽  
Florencia Palacios ◽  
Wentian Li ◽  
Sophia Yancopoulos ◽  
Carlo Calissano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Antigen Presentation ◽  
B Lymphocytes ◽  
Research Funding ◽  
Myeloid Cell ◽  
Integrin Signaling ◽  
Gene Sets

Abstract CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of > 1.5 or < -1.5 and a level of significance of P < 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.

BAFF-R Receptor Also Functions in the Nucleus of Normal and Neoplastic Human B Lymphocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2368-2368
Author(s):  
Lingchen Fu ◽  
Yen-Chiu Lin-Lee ◽  
Archito Tamayo ◽  
Linda Yoshimura ◽  
Richard J. Ford
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
B Lymphocytes ◽  
Cell Nucleus ◽  
Peripheral Blood ◽  
Target Genes ◽  
B Lymphocyte ◽  
Normal Peripheral Blood

Abstract B-lymphocyte stimulator (BLyS) is a relatively newly described tumor necrosis factor (TNF) superfamily cytokine involved in cell survival and proliferation in normal and neoplastic B cells, particularly in the aggressive B cell non-Hodgkin’s lymphomas (NHL-B). BLyS binds to three receptors: BAFF-R (or BR3), BCMA and TACI. However, recent studies have shown that BLyS regulates B cell survival and proliferation predominately through BAFF-R. Mice with mutant BAFF-R show significant decrease in the peripheral blood B-lymphocyte compartment, similar to the phenotype of BLyS knockout mice. BLyS/BAFF-R signaling functions through activation of the critical transcription factor NF-kB pathways, especially the NF-kB2 pathway, which mainly involves the p52/ rel-B complex. Our previous studies have shown that BLyS is constitutively expressed in aggressive NHL-B cells leading to increased survival and proliferation of the malignant B cells. In this study, we found by western blotting and confocal microscopy that BAFF-R, the major B cell associated cell membrane receptor for BLyS, was also present in the B cell nucleus, in addition to its location in plasma membrane and cytoplasm in both normal peripheral blood B lymphocytes and NHL-B cells. Nuclear presence can be increased by anti-IgM and soluble CD154 treatment in normal peripheral blood B lymphocytes, and is constitutively expressed in aggressive NHL-B. Inhibition of BLyS expression by specific BLyS siRNA decreases nuclear BAFF-R level and survival in LBCL cells. Immunostaining experiments show that in the cytoplasm of normal and neoplastic B cells, BAFF-R interacts with the improtin a and b which are members of the classic karypherin pathway. A candidate nuclear localization sequence (NLS) was also identified in the BAFF-R protein sequence, and mutation of this putative NLS can block BAFF-R entering nucleus, which is consistent with our hypothesis that BAFF-R undergoes nuclear translocation. To further investigate the functional role of BAFF-R in nucleus, we performed confocal immunostaining analysis and co-immunoprecipitation, that shows that BAFF-R co-localizes with some NF-kB family members such as c-rel in the B cell nucleus. We also found that nuclear BAFF-R/c-rel complex can bind to the NF-kB binding site on the promoters of NF-kB target genes such as BLyS, CD154, Bcl-xL and Bfl-1 /A1 by chromatin immnoprecipitation (ChIP) assay. Luciferase reporter assays show that BAFF-R has transactivation activity on these NF-kB target genes. Furthermore, NLS mutant BAFF-R decreases NF-kB target gene promoter activity, compared to wild type BAFF-R, and NHL-B cell proliferation. These findings indicates that in addition to activating NF-kB pathways at the plasma membrane, BAFF-R may also promote survival and proliferation of both normal and NHL-B cell in the nucleus by directly regulating transcriptional activity of key NF-kB target genes and may functions as a transcriptional co- factor with NF-kB.

Sign in / Sign up

Export Citation Format

Share Document