scholarly journals Nonpermissive bone marrow environment impairs early B-cell development in common variable immunodeficiency

Blood ◽  
2020 ◽  
Vol 135 (17) ◽  
pp. 1452-1457 ◽  
Author(s):  
Arianna Troilo ◽  
Claudia Wehr ◽  
Iga Janowska ◽  
Nils Venhoff ◽  
Jens Thiel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Immature B Cells ◽  
Cell Repopulation

Abstract Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

scholarly journals The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 79-79
Author(s):  
Zev J. Greenberg ◽  
Darlene A. Monlish ◽  
Rachel L. Bartnett ◽  
Jeffrey J. Bednarski ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Development ◽  
B Cell Development ◽  
Cellular Migration ◽  
Physical Interaction ◽  
Hematopoietic Stem ◽  
Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

scholarly journals Developmental Stage Specific B-Progenitor Expansion in Normal Fetal Bone Marrow Is Absent in Down Syndrome: Implications for Infant ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4331-4331
Author(s):  
Anindita Roy ◽  
Georg Bohn ◽  
Katerina Goudevenou ◽  
Gillian Cowan ◽  
Neha Bhatnagar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Fetal Life ◽  
Second Trimester ◽  
Leukemic Transformation ◽  
Developmentally Regulated ◽  
Cd34 Cells

Abstract Although distinct fetal and adult B-cell lineage development has been demonstrated in murine studies, human fetal B-lymphopoiesis remains poorly understood. Previous work from our lab identified a population of PreProB progenitors (CD34+CD19+CD10-) in fetal liver (FL), co-existing with adult-type CD34+CD19+CD10+ ProB progenitors (Roy, A., Cowan G et al. PNAS: 109, 17579-17584; 2012). This supports other work demonstrating a PreProB progenitor population in normal cord blood (Sanz, E., et al. PNAS: 107, 5925-5930; 2010) and suggests that fetal pathways of B-cell development are also likely to be markedly different from adult bone marrow (AdBM) in humans. FL PreProB progenitors give rise solely to B-cells in vitro and display a gene expression pattern of successive activation of B-cell transcription factors as determined by Fluidigm RQ-PCR. In addition, increasing evidence indicates that infant ALL and many cases of childhood ALL arise in fetal life, suggesting that ontogeny-related changes in B-cell development may provide the cellular and microenvironmental context for in utero leukemia initiation. We therefore investigated B-cell development in normal human fetal BM from the onset of BM hematopoiesis in late first trimester. The composition and function of the early lymphoid and committed B-progenitor compartments of fetal BM(12-22 weeks; n=20) were compared with FL (n=25) at the same gestation, paediatric (Paed) BM (n=6) and AdBM (n=7), by multiparameter flow cytometry, differentiation in stromal co-culture assays and clonogenic assays. All stages of B-cell development were demonstrable in human fetal BM. However, there was a significantly higher frequency of B-progenitors in fetal BM (45.8±2.7% of CD34+ cells) compared to FL (10.3±0.97%; p<0.0001), PaedBM (28.2±4.2%; p=0.001) and AdBM (25.8±2.8%; p<0.0001). As in FL, both CD34+CD19+CD10- (PreProB) and CD34+CD19+10+ (ProB) progenitors were identified in fetal BM. PreProB progenitors were significantly higher in fetal BM (21.9±2.3% of CD34+ cells) compared to FL (3.8±0.4%), PaedBM (4.2±0.9%) and AdBM (3.4±0.9%) (p<0.0001 for all). Fetal BM PreProB progenitors gave rise solely to B-cells when co-cultured on MS5 stromal cells with FLT3L, SCF and IL7 in contrast to multilineage output of fetal BM HSC and LMPP cultured under identical conditions. Furthermore, fetal BM Lin-CD10-CD34+ cells cultured in vitro acquire CD19 expression before CD10 expression (n=6) consistent with a unique, fetal-specific B-cell differentiation pathway. The progressive decline in the proportion of PreProB progenitors from 47.8% to 36.8%, 14.8% and 13% of total B-progenitors in fetal BM, FL, PaedBM and AdBM respectively points towards a developmental stage-specific emergence of these progenitors. Finally, the fetal BM B-progenitor: B-cell ratio falls rapidly from ~4:1 at 12 weeks gestation to 1:1 at 18 weeks gestation as mature B-cell production in fetal BM gradually increases. This is consistent with a developmentally-regulated drive to B-progenitor proliferation, at the expense of differentiation, early in the second trimester which might represent a target population vulnerable to leukemic transformation in fetal life. Since children with Down syndrome (DS) do not develop infant ALL, we examined DS fetal BM as they may lack susceptible target cells for leukemic transformation. Consistent with this, PreProB progenitors in DS fetal BM (n=7) were >6-fold lower than normal fetal BM (3.3±0.8% vs. 21.9±2.3% of CD34+ cells; p<0.0001). Furthermore, CD19+ cells with an infant ALL-associated immunophenotype (CD19+CD10-CD20-) are detectable in normal fetal BM mononuclear cells, but are rarely found in DS fetal BM. Conclusion: There is a marked expansion of PreProB progenitors in normal second trimester human fetal BM which is virtually absent in DS fetal BM, in normal PaedBM and in normal AdBM. We suggest that developmentally-regulated, functional and molecular characteristics of these fetal-specific B-progenitors may provide the 'oncogenic' cellular context necessary to co-operate with genetic events, such as MLL rearrangements, to induce ALL in infants without DS. Disclosures No relevant conflicts of interest to declare.

B Cell Development in the Absence of PU.1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 226-226 ◽  
Author(s):  
Min Ye ◽  
Olga Ermaermakova-Cirilli ◽  
Thomas Graf
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Fetal Liver ◽  
Cell Formation ◽  
Cell Development ◽  
B Cell Development ◽  
B Lineage

Abstract Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.

Absence of the Wnt Antagonist Sclerostin Adversely Affects B Cell Development in the Bone Marrow Niche

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 220-220 ◽  
Author(s):  
Corey J Cain ◽  
Randell Rueda ◽  
Bryce T McLelland ◽  
Nicole Collette ◽  
Gabriela Loots ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Wnt Signaling ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Osteoblast Activity

Abstract Abstract 220 Hematopoietic cell fate decisions are dependent on their localized microenvironmental niche. In the bone, endosteal osteoblasts have been shown to support hematopoietic stem cells (HSC) self-renewal, as demonstrated by transgenic and knockout mouse models in which osteoblast populations were increased or decreased. In addition, Wnt signaling and the Wnt antagonist Dkk-1 have been implicated in various aspects of hematopoiesis and HSC self-renewal. Sclerostin (Sost) is a secreted protein that is primarily expressed by fully mature osteocytes and acts on osteoblasts as a negative regulator of bone growth, by antagonizing Wnt signaling by its binding to the Wnt co-receptors Lrp4, Lrp5, and/or Lrp6. Here, we investigated the role of Sost on hematopoiesis in the bone marrow niche. Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in hypermineralized bones with small bone marrow cavities. As such, Sost−/− mice contain markedly reduced numbers of CD45+hematopoietic cells in the bone marrow. Since hematopoietic stem cell activity is dependent on osteoblast function, we examined whether the hyperactive osteoblast activity in Sost−/− mice influences the numbers of hematopoietic stem cells, lymphoid progenitor cells and myeloid progenitor cells in the bone marrow. Surprisingly, no differences were observed in hematopoietic stem and progenitor cell frequency and cell number. However, we found the bone marrow of Sost−/− mice to be depleted of B cells, and this reduction can be attributed to premature apoptosis beginning at the pre-pro-B cell stage. Examination of Sost expression showed that no hematopoietic cells expressed Sost, however, pre-pro, immature and recirculating B cells expressed Lrp5 and Lrp6. These gene expression patterns suggested that the defect in B cell development in Sost−/− mice is non-cell autonomous and that absence of Sost could affect Wnt signaling in these populations. We observed that the expression of Wnt target genes CCND1 and Lef-1 were not affected by the absence of Sost, but c-Myc was significantly upregulated in recirculating B cells in the bone marrow. We also observed a significant decrease in CXCL12 expression in the bone marrow stroma in Sost−/− mice, consistent with their inability to adequately support B cell development. Taken together, our results indicate that the B cell developmental defects in Sost−/− mice are non-cell autonomous, and we are currently performing reciprocal bone marrow transplantation experiments to further support this hypothesis. Our studies demonstrate a novel role for Sost in the regulation of B cell development in the bone marrow, and demonstrate that distinct Wnt antagonists play specific roles in the regulation of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1465-1465
Author(s):  
Jason Mullenix ◽  
Kimi Y Kong ◽  
Kristin Severns Owens ◽  
Jason Rogers ◽  
Shannon FitzPatrick ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mature Mirnas ◽  
Cell Development ◽  
B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 518-525 ◽  
Author(s):  
Shunichi Takeda ◽  
Takeyuki Shimizu ◽  
Hans-Reimer Rodewald
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

scholarly journals Sclerostin Deficiency Alters Peripheral B Lymphocyte Responses in Mice

2018 ◽  
Author(s):  
Arthur Chow ◽  
Jourdan Mason ◽  
Larrisha Coney ◽  
Jamila Bajwa ◽  
Cameron Carlisle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
Protein Antigens ◽  
Hematopoietic Stem ◽  
B Cell Response

AbstractUnderstanding how changes in bone physiology and homeostasis affect immune responses will inform how to retain strong immunity in patients with bone disease and in aged individuals. We previously identified sclerostin (Sost) as a mediator of cell communication between the skeletal and the immune system. Elevated bone mineral density in Sost-knockout (Sost-/-) mice contributes to an altered bone marrow microenvironment and adversely affects B cell development. B cells originate from hematopoietic stem cells within the bone marrow and mature in peripheral lymphoid organs to produce antibodies in response to infection and/or vaccination. In this study, we investigated whether the aberrant B cell development observed in the bone marrow of Sost-/- mice extends to peripheral B cells in the spleen during immune challenge, and if these changes were age-dependent. Concomitant with more severe changes in bone architecture, B cell development in the bone marrow and in the spleen worsened with age in Sost-/- mice. B cell responses to T-independent antigens were enhanced in young Sost-/- mice, whereas responses to T-dependent antigens were impaired. Our results support the hypothesis that the adverse effects of B cell development in the Sost-deficient bone marrow microenvironment extends to the peripheral B cell immune response to protein antigens, and suggest that the B cell response to routine vaccinations should be monitored regularly in patients being treated with sclerostin antibody therapy. In addition, our results open the possibility that Sost regulates the T-independent B cell response, which might be applicable to the improvement of vaccines towards non-protein antigens.

scholarly journals Ras activation of Erk restores impaired tonic BCR signaling and rescues immature B cell differentiation

2010 ◽  
Vol 207 (3) ◽  
pp. 607-621 ◽  
Author(s):  
Sarah L. Rowland ◽  
Corinne L. DePersis ◽  
Raul M. Torres ◽  
Roberta Pelanda
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Constitutive Expression ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
Bcr Signaling ◽  
Immature B Cells

B cell receptors (BCRs) generate tonic signals critical for B cell survival and early B cell development. To determine whether these signals also mediate the development of transitional and mature B cells, we examined B cell development using a mouse strain in which nonautoreactive immunoglobulin heavy and light chain–targeted B cells express low surface BCR levels. We found that reduced BCR expression translated into diminished tonic BCR signals that strongly impaired the development of transitional and mature B cells. Constitutive expression of Bcl-2 did not rescue the differentiation of BCR-low B cells, suggesting that this defect was not related to decreased cell survival. In contrast, activation of the Ras pathway rescued the differentiation of BCR-low immature B cells both in vitro and in vivo, whereas extracellular signal-regulated kinase (Erk) inhibition impaired the differentiation of normal immature B cells. These results strongly suggest that tonic BCR signaling mediates the differentiation of immature into transitional and mature B cells via activation of Erk, likely through a pathway requiring Ras.

The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2465-2465
Author(s):  
Wanda P. Blanton ◽  
Fangnian Wang ◽  
Hongsheng Liu ◽  
Paul Romesser ◽  
Douglas Faller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Brdu Incorporation ◽  
Cell Compartment ◽  
Hematopoietic Stem

Abstract Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.

Single Cell Trajectory Detection Orders Hallmarks of Early Human B Cell Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1044-1044 ◽  
Author(s):  
Kara L. Davis ◽  
Sean C Bendall ◽  
El-ad D. Amir ◽  
Erin F. Simonds ◽  
Astraea Jager ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Developmental Process ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Hematopoietic Stem ◽  
Human B Cell

Abstract Abstract 1044 Background: Normal B cell development in the bone marrow (BM) is a seemingly well-understood, progressive process and thus represents a suitable test system in which to apply an algorithmic approach to modeling cellular differentiation. In humans, hematopoietic stem cells form lymphoid progenitor cells that develop into pro- then pre- B cells and finally those cells that escape negative selection become immature B cells that leave the BM for the peripheral immune organs. Flow cytometry can track these stages using the expression of immunophenotypic cell surface markers, including those for progenitors (CD34, CD38), early B cell populations (CD10), as well as those of more mature B cells (CD20, IgM). Expression of the B cell transcription factor PAX5, and immune diversity conferring enzymes terminal deoxynucleotidyl transferase (TdT) and recombination-activating gene (RAG) can also be tracked at the single cell level. Regulatory signaling by factors in the BM orchestrates critical checkpoints in the B cell developmental program, such as Interleukin (IL)-7-mediated STAT5 phosphorylation and signaling downstream the preB cell receptor/B cell receptor (BCR) (p-BLNK, p-Syk, p-PLCγ2, p-Erk). Successful coordination of these signals with immunoglobulin gene rearrangement results in a burst of proliferative expansion prior to maturation/exit to the periphery. Failure of any one of these processes results in B cell deletion while certain dysregulations driven by oncogenic processes can result in malignancy. While much of this core understanding has been founded in murine models, the rarity of early B cell progenitors and lack of genetic tools has complicated definition of B cell development in humans. Using 42 parameter mass cytometry in combination with a novel single-cell trajectory finding algorithm, we have now laid a human B cell developmental process in primary human BM to an unprecedented level of detail, mapping out the expression pattern of virtually all relevant B cell immunophenotypic markers as well as intracellular enzyme, transcription factor and regulatory modification simultaneously, at the single cell level. Methods: The mononuclear cell fraction of multiple healthy human marrows was characterized by simultaneously analyzing 42 antibody parameters with mass cytometry targeting a multitude of phenotypic markers, intracellular signaling molecules, hallmarks of cell cycle and apoptosis all in the context of in vitro perturbations relevant to B cell development (including IL-7 and BCR crosslinking). The resulting multidimensional data was modeled using a novel, scalable, robust graph-based trajectory algorithm that iteratively refines a solution trajectory using random landmarks to reduce variability. Populations of interest were prospectively isolated and a novel qPCR assay was created to quantitate immunoglobulin heavy chain (IgH) rearrangement in genomic DNA. Results/Conclusion: Modeling of the resulting data was undertaken using this algorithm (termed Wanderlust) that devised and ordered cellular relationships based on the average phenotypic progression from our defined starting point, in this case, CD34+CD38- hematopoietic stem cells, in order to calculate a developmental trajectory. The predicted trajectory was then used to inform a traditional 'gating' analysis of the data and provide a higher resolution view of human B cell development than previously published. It both confirmed established steps in human B cell progression, and importantly, revealed new populations of early B cell progenitors based on expression of CD34, CD38, CD24 and TdT. These populations were corroborated to be of B-lineage and ordered as predicted based on the progressive rearrangement of the IgH locus by qPCR of extracted genomic DNA. We aligned previously unregistered key developmental checkpoints such as STAT5 activation in response to IL-7 and proliferation in response preBCR expression with traditional immunophenotypic cell populations. While predicted in silico, and then molecularly verified and staged in vitro, these regulatory events all lay within discrete cell subsets that can now be demarcated using conventional cytometric methods. Together, this provides a backbone on which to further examine both healthy regulatory events as well as the corruption of this developmental process such as in malignant or immunodeficient states. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document