Human clones with natural killer function can activate B cells and secrete B cell differentiation factors

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.

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Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3929 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3929-3936 ◽

Cited By ~ 16

Author(s):

T D Randall ◽

F E Lund ◽

J W Brewer ◽

C Aldridge ◽

R Wall ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

B Cell Lymphoma ◽

Receptor Expression ◽

High Rate ◽

B Cell Differentiation ◽

Interleukin 5 ◽

Stage Of Development ◽

Differentiation Factors

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.

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AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3507 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1372-1373

Author(s):

G. M. Verstappen ◽

J. C. Tempany ◽

H. Cheon ◽

A. Farchione ◽

S. Downie-Doyle ◽

...

Keyword(s):

Cell Division ◽

Cell Differentiation ◽

T Cell ◽

B Cells ◽

B Cell ◽

B Cell Differentiation ◽

Research Support ◽

Differentiation Capacity ◽

Cell Responses ◽

Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

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YY1 plays an essential role at all stages of B-cell differentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606297113 ◽

2016 ◽

Vol 113 (27) ◽

pp. E3911-E3920 ◽

Cited By ~ 43

Author(s):

Eden Kleiman ◽

Haiqun Jia ◽

Salvatore Loguercio ◽

Andrew I. Su ◽

Ann J. Feeney

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Signaling Pathways ◽

Mrna Splicing ◽

Cell Populations ◽

Sequencing Analysis ◽

B Cell Differentiation ◽

Chip Sequencing

Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro–B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.

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Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

10.20944/preprints202111.0365.v1 ◽

2021 ◽

Author(s):

Casper Marsman ◽

Dorit Verhoeven

Keyword(s):

Cell Differentiation ◽

T Cell ◽

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Formation ◽

B Cell Differentiation ◽

Differentiation Potential ◽

Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

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Conserved gammaherpesvirus protein kinase counters the antiviral effects of myeloid cell specific STAT1 expression to promote the establishment of splenic B cell latency.

Journal of Virology ◽

10.1128/jvi.00859-21 ◽

2021 ◽

Author(s):

P. A. Sylvester ◽

C. N. Jondle ◽

K. P. Stoltz ◽

J. Lanham ◽

B. N. Dittel ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

Protein Kinase ◽

B Cell ◽

Latent Reservoir ◽

Type I ◽

B Cell Differentiation ◽

Cell Type ◽

Antiviral Effects ◽

Cell Type Specific

Gammaherpesviruses establish life-long infections and are associated with B cell lymphomas. Murine gammaherpesvirus-68 (MHV68) infects epithelial and myeloid cells during acute infection, with subsequent passage of the virus to B cells, where physiological B cell differentiation is usurped to ensure the establishment of chronic latent reservoir. Interferons (IFNs) represent a major antiviral defense system that engages transcriptional factor STAT1 to attenuate diverse acute and chronic viral infections, including those of gammaherpesviruses. Correspondingly, global deficiency of type I or type II IFN signaling profoundly increases the pathogenesis of acute and chronic gammaherpesvirus infection, compromises host survival, and impedes mechanistic understanding of cell type-specific role of IFN signaling. Here we demonstrate that myeloid-specific STAT1 deficiency attenuates acute and persistent MHV68 replication in the lungs and suppresses viral reactivation from peritoneal cells, without any effect on the establishment of viral latent reservoir in splenic B cells. All gammaherpesviruses encode a conserved protein kinase that antagonizes type I IFN signaling in vitro. Here, we show that myeloid-specific STAT1 deficiency rescues the attenuated splenic latent reservoir of kinase null MHV68 mutant. However, despite having gained access to splenic B cells, protein kinase null MHV68 mutant fails to drive B cell differentiation. Thus, while myeloid-intrinsic STAT1 expression must be counteracted by the gammaherpesvirus protein kinase to facilitate viral passage to splenic B cells, expression of the viral protein kinase continues to be required to promote optimal B cell differentiation and viral reactivation, highlighting the multifunctional nature of this conserved viral protein during chronic infection. Importance. IFN signaling is a major antiviral system of the host that suppresses replication of diverse viruses, including acute and chronic gammaherpesvirus infection. STAT1 is a critical member and the primary antiviral effector of IFN signaling pathways. Given the significantly compromised antiviral status of global type I or type II IFN deficiency, unabated gammaherpesvirus replication and pathogenesis hinders understanding of cell type-specific antiviral effects. In this study, a mouse model of myeloid-specific STAT1 deficiency unveiled site-specific antiviral effects of STAT1 in the lungs and peritoneal cavity, but not spleen of chronically infected hosts. Interestingly, expression of a conserved gammaherpesvirus protein kinase was required to counteract the antiviral effects of myeloid-specific STAT1 expression to facilitate latent infection of splenic B cells, revealing a cell-type specific virus-host antagonism during the establishment of chronic gammaherpesvirus infection.

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Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid

Blood ◽

10.1182/blood.v83.8.2206.2206 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2206-2210 ◽

Cited By ~ 6

Author(s):

Y Levy ◽

S Labaume ◽

MC Gendron ◽

JC Brouet

Keyword(s):

Retinoic Acid ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Plasma Cells ◽

B Cell Differentiation ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Culture Supernatants

Abstract We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

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Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

Blood ◽

10.1182/blood.v96.7.2338 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2338-2345 ◽

Cited By ~ 115

Author(s):

Roman Krzysiek ◽

Eric A. Lefevre ◽

Jérôme Bernard ◽

Arnaud Foussat ◽

Pierre Galanaud ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Chemokine Receptor ◽

Memory B Cells ◽

Receptor Expression ◽

B Cell Differentiation ◽

Hematopoietic Stem ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein

Abstract The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

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B-cell differentiation following autologous, conventional, or T-cell depleted bone marrow transplantation: a recapitulation of normal B-cell ontogeny

Blood ◽

10.1182/blood.v76.8.1647.1647 ◽

1990 ◽

Vol 76 (8) ◽

pp. 1647-1656 ◽

Cited By ~ 105

Author(s):

TN Small ◽

CA Keever ◽

S Weiner-Fedus ◽

G Heller ◽

RJ O'Reilly ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

Bone Marrow Transplantation ◽

B Cells ◽

Cord Blood ◽

B Cell ◽

First Year ◽

B Cell Differentiation ◽

And Function ◽

Cell Ontogeny

Abstract The circulating lymphocytes of 88 consecutive patients following autologous, conventional, or T-cell depleted bone marrow transplantation were serially analyzed for B-cell surface antigen expression and function. In the majority of patients, except for those who developed chronic graft-versus-host disease, the number of circulating CD20+ B cell normalized by the fourth posttransplant month. The earliest detectable B cells normally expressed HLA-DR, CD19, surface immunoglobulin (slg), CD21, Leu-8, and lacked expression of CD10 (CALLA). In addition, the circulating B cells expressed CD1c, CD38, CD5, and CD23 for the first year following transplant, antigens that are normally expressed on a small percentage of circulating B cells in normal adults, but highly expressed on cord blood B cells. Similar to cord blood B cells, patient B cells isolated during the first year following transplant, proliferated normally to Staphylococcus aureus Cowan strain I (SAC), and produced IgM, but minimal or no IgG when stimulated with pokeweed mitogen and SAC, unlike normal adult B cells that produce both. The similar phenotype and function of posttransplant and cord blood B cells, and their similar rate of decline in patients and normal children adds further evidence to support the hypothesis that B-cell differentiation posttransplant is recapitulating normal B-cell ontogeny.

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Identification of a B cell differentiation factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the lpr/lpr genotype.

Journal of Experimental Medicine ◽

10.1084/jem.157.2.730 ◽

1983 ◽

Vol 157 (2) ◽

pp. 730-742 ◽

Cited By ~ 121

Author(s):

G J Prud'Homme ◽

C L Park ◽

T M Fieser ◽

R Kofler ◽

F J Dixon ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Mouse Strain ◽

Interleukin 2 ◽

Lymphoid Cells ◽

B Cell Differentiation ◽

Differentiation Factor ◽

Cell Differentiation Factor

Lymph node and spleen cells of the autoimmune MRL/Mp-lpr/lpr mouse strain spontaneously produce (in the absence of mitogenic stimulation) a factor(s) that induces B cell differentiation. This factor is not produced by the congenic MRL/n mouse strain that lacks the lpr gene or by normal mouse strains. However, lymphoid cells of the B6-lpr/lpr (B6/1) strain also produce a B cell differentiation factor. Although the factor acts on resting B cells, its effect is greatly magnified by activating the B cells with anti-mu or lipopolysaccharide. MRL/l mice begin producing the factor as early as 1 mo of age but levels increase with age and appearance of lymphoproliferation. Cell depletion studies reveal that this factor is produced by T cells of the Lyt-1+2-phenotype. Because of its association with the lpr/lpr genotype, we term this B cell differentiation factor L-BCDF. Functional analysis of L-BCDF reveals that it acts regardless of cell density in culture and in the absence of interleukin 2 (IL-2). In fact, the increase in the production of L-BCDF by MRL/1 T cells with aging occurs concomitantly with a marked decrease in their ability to produce IL-2. No T cell replacing factor activity or B cell growth factor-like activity can be detected in MRL/l-derived supernatants. L-BCDF induces both IgM and IgG synthesis in lipopolysaccharide-activated B cells; however, it has a greater effect on IgG secretion. In particular, the production of IgG1, IgG2a, and IgG2b are markedly enhanced in the presence of L-BCDF. The spontaneous production of L-BCDF by T cells of SLE mice of lpr/lpr genotype suggests an association of this factor with autoimmunity.

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