B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats

Intra-renal tertiary lymphoid organs (TLOs) are associated with worsened outcome in kidney transplantation (Ktx). We used an anti-BAFF (B cell activating factor) intervention to investigate whether BAFF is required for TLO formation in a full MHC-mismatch Ktx model in rats. Rats received either therapeutic immunosuppression (no rejection, NR) or subtherapeutic immunosuppression (chronic rejection, CR) and were sacrificed on d56. One group additionally received an anti-BAFF antibody (CR + AB). Intra-renal T (CD3+) and B (CD20+) cells, their proliferation (Ki67+), and IgG+ plasma cells were analyzed by immunofluorescence microscopy. Formation of T and B cell zones and TLOs was assessed. Intra-renal expression of TLO-promoting factors, molecules of T:B crosstalk, and B cell differentiation was analyzed by qPCR. Intra-renal B and T cell zones and TLOs were detected in CR and were associated with elevated intra-renal mRNA expression of TLO-promoting factors, including CXCL13, CCL19, lymphotoxin-β, and BAFF. Intra-renal plasma cells were also elevated in CR. Anti-BAFF treatment significantly decreased intra-renal B cell zones and TLO, as well as intra-renal B cell-derived TLO-promoting factors and B cell differentiation markers. We conclude that BAFF-dependent intra-renal B cells promote TLO formation and advance local adaptive alloimmune responses in chronic rejection.

Download Full-text

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.

Download Full-text

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.

Download Full-text

B Cell Activating Factor Inhibition Impairs Bacterial Immunity by Reducing T Cell-Independent IgM Secretion

Infection and Immunity ◽

10.1128/iai.00998-13 ◽

2013 ◽

Vol 81 (12) ◽

pp. 4490-4497 ◽

Cited By ~ 9

Author(s):

Derek D. Jones ◽

Maura Jones ◽

Gregory A. DeIulio ◽

Rachael Racine ◽

Katherine C. MacNamara ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

B Cell ◽

Host Defense ◽

B Cell Differentiation ◽

Content Type ◽

Surface Marker Expression ◽

B Cell Activating Factor

ABSTRACTB cell activating factor of the tumor necrosis factor family (BAFF) is an essential survival factor for B cells and has been shown to regulate T cell-independent (TI) IgM production. DuringEhrlichia murisinfection, TI IgM secretion in the spleen was BAFF dependent, and antibody-mediated BAFF neutralization led to an impairment of IgM-mediated host defense. The failure of TI plasmablasts to secrete IgM was not a consequence of alterations in their generation, survival, or early differentiation, since all occurred normally in infected mice following BAFF neutralization. Gene expression characteristic of plasma cell differentiation was also unaffected by BAFF neutralizationin vivo, and except for CD138, plasmablast cell surface marker expression was unaffected. IgM was produced, since it was detected intracellularly, and impaired secretion was not due to a failure to express the IgM secretory exon. Addition of BAFF to plasmablastsin vitrorescued IgM secretion, suggesting that BAFF signaling can directly regulate secretory processes. Our findings indicate that BAFF signaling can modulate TI host defense by acting at a late stage in B cell differentiation, via its regulation of terminal plasmablast differentiation and/or IgM secretion.

Download Full-text

The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity

Journal of Experimental Medicine ◽

10.1084/jem.20131831 ◽

2014 ◽

Vol 211 (5) ◽

pp. 827-840 ◽

Cited By ~ 52

Author(s):

Stéphane Chevrier ◽

Dianne Emslie ◽

Wei Shi ◽

Tobias Kratina ◽

Cameron Wellard ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Cell Cycle Progression ◽

Plasma Cells ◽

Cell Lineage ◽

B Cell Differentiation ◽

Antibody Secreting Cell ◽

Important Player

The transcriptional network regulating antibody-secreting cell (ASC) differentiation has been extensively studied, but our current understanding is limited. The mechanisms of action of known “master” regulators are still unclear, while the participation of new factors is being revealed. Here, we identify Zbtb20, a Bcl6 homologue, as a novel regulator of late B cell development. Within the B cell lineage, Zbtb20 is specifically expressed in B1 and germinal center B cells and peaks in long-lived bone marrow (BM) ASCs. Unlike Bcl6, an inhibitor of ASC differentiation, ectopic Zbtb20 expression in primary B cells facilitates terminal B cell differentiation to ASCs. In plasma cell lines, Zbtb20 induces cell survival and blocks cell cycle progression. Immunized Zbtb20-deficient mice exhibit curtailed humoral responses and accelerated loss of antigen-specific plasma cells, specifically from the BM pool. Strikingly, Zbtb20 induction does not require Blimp1 but depends directly on Irf4, acting at a newly identified Zbtb20 promoter in ASCs. These results identify Zbtb20 as an important player in late B cell differentiation and provide new insights into this complex process.

Download Full-text

Increased Frequency of Pre–Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for aDevelopmental Block in B Cell Differentiation

Developmental Immunology ◽

10.1080/1044667021000003961 ◽

2002 ◽

Vol 9 (1) ◽

pp. 35-45 ◽

Cited By ~ 7

Author(s):

Zhe-Xiong Lian ◽

Hiroto Kita ◽

Tomoyuki Okada ◽

Tom Hsu ◽

Leonard D. Shultz ◽

...

Keyword(s):

New Zealand ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Cell Differentiation ◽

Differentiation Markers ◽

Cell Stage

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor componentIgα(mb-1). Furthermore, levels of expression of theRug2, λ5andIgβ(B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

Download Full-text

Chromatin reader Dido3 regulates the genetic network of B cell differentiation

10.1101/2021.02.23.432411 ◽

2021 ◽

Author(s):

Fernando Gutiérrez del Burgo ◽

Tirso Pons ◽

Enrique Vázquez de Luis ◽

Carlos Martínez-A ◽

Ricardo Villares

Keyword(s):

Transcription Factors ◽

Cell Differentiation ◽

B Cell ◽

Cell Lineage ◽

Genetic Network ◽

B Cell Differentiation ◽

Differentiation Markers ◽

Hematopoietic Stem ◽

Severe Deficiency ◽

The Impact

ABSTRACTThe development of hematopoietic lineages is based on a complex balance of transcription factors whose expression depends on the epigenetic signatures that characterize each differentiation step. The B cell lineage arises from hematopoietic stem cells through the stepwise silencing of stemness genes and balanced expression of mutually regulated transcription factors, as well as DNA rearrangement. Here we report the impact on B cell differentiation of the lack of DIDO3, a reader of chromatin status, in the mouse hematopoietic compartment. We found reduced DNA accessibility in hematopoietic precursors, leading to severe deficiency in the generation of successive B cell differentiation stages. The expression of essential transcription factors and differentiation markers is affected, as is the somatic recombination process.One Sentence Summary: Epigenetic control of early hematopoiesis

Download Full-text

TLR-mediated activation of Waldenström macroglobulinemia B cells reveals an uncoupling from plasma cell differentiation

Blood Advances ◽

10.1182/bloodadvances.2019001279 ◽

2020 ◽

Vol 4 (12) ◽

pp. 2821-2836

Author(s):

Jennifer Shrimpton ◽

Matthew A. Care ◽

Jonathan Carmichael ◽

Kieran Walker ◽

Paul Evans ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Plasma Cells ◽

Immunoglobulin M ◽

B Cell Differentiation ◽

Waldenström Macroglobulinemia ◽

Plasma Cell Differentiation ◽

Waldenstrom Macroglobulinemia

Abstract Waldenström macroglobulinemia (WM) is a rare malignancy in which clonal B cells infiltrate the bone marrow and give rise to a smaller compartment of neoplastic plasma cells that secrete monoclonal immunoglobulin M paraprotein. Recent studies into underlying mutations in WM have enabled a much greater insight into the pathogenesis of this lymphoma. However, there is considerably less characterization of the way in which WM B cells differentiate and how they respond to immune stimuli. In this study, we assess WM B-cell differentiation using an established in vitro model system. Using T-cell–dependent conditions, we obtained CD138+ plasma cells from WM samples with a frequency similar to experiments performed with B cells from normal donors. Unexpectedly, a proportion of the WM B cells failed to upregulate CD38, a surface marker that is normally associated with plasmablast transition and maintained as the cells proceed with differentiation. In normal B cells, concomitant Toll-like receptor 7 (TLR7) activation and B-cell receptor cross-linking drives proliferation, followed by differentiation at similar efficiency to CD40-mediated stimulation. In contrast, we found that, upon stimulation with TLR7 agonist R848, WM B cells failed to execute the appropriate changes in transcriptional regulators, identifying an uncoupling of TLR signaling from the plasma cell differentiation program. Provision of CD40L was sufficient to overcome this defect. Thus, the limited clonotypic WM plasma cell differentiation observed in vivo may result from a strict requirement for integrated activation.

Download Full-text