Protein Signature of LMP1 Signaling in PTLDs Identifies and Mimics Inter/Perifollicular CD30+ EBV− B Blasts.

Abstract EBV-associated B-cell Post-Transpant Lymphoproliferative Disorders (PTLDs) represent a diverse group of lesions morphologically, in clinical presentation and behaviour, ranging from early reversible lesions to monomorphic aggressive lymphomas. Polymorphic cases, which represent the focus of our analysis, contain a mixture of cells in various EBV latency stages, defined by EBNA1, EBNA2 and LMP1 immunostaining. LMP1 is a key viral protein for cellular transformation and, analogously to CD40, engages TNF Receptor Associated Proteins and activates NF-kB and NF-kB-responsive genes. We analyzed the protein signature of LMP1 in PTLDs and non-PTLD tonsils by double staining for LMP1, CD30, CD20, Pax5 and signaling molecules. A remarkably conserved set of proteins, associated with LMP1/CD40 signaling and NF-kB activation is expressed both in the EBV-infected lymphoid population in polymorphic PTLDs and in a normal B-cell subset(s) in reactive tonsils. These proteins include highly expressed CD30, JunB, nuclear cRel, TRAF-1, Bcl-XL, MUM1, CCL22 and downregulated BCL6 and CD10. We observed that EBV infection, possibly through LMP1 and LMP2A signaling, results in varioius degrees of differentiation within the neoplastic clone. EBER+ terminally differentiated mucosa-associated IRTA-1+ marginal zone B-cells and CD138+ plasma cells were identified in most cases, including control post-transplant tonsils with no overt disease. We document for the first time in situ, in-vivo evidence of EBV latently infected post-Germinal Center B cells of marginal and plasma cell types in PTLDs. Polymorphic PTLD cases represent EBV-induced expansion of B cells, mimicking CD40L-like activated Peri/Interfollicular CD30+ normal B-cells.

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The transcription factor Zeb2 drives differentiation of age-associated B cells

10.1101/2021.07.24.453633 ◽

2021 ◽

Author(s):

Shuangshuang Gu ◽

Xiaxia Han ◽

Chao Yao ◽

Huihua Ding ◽

Jinsong Zhang ◽

...

Keyword(s):

Transcription Factor ◽

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Subset ◽

Signature Genes ◽

Systemic Autoimmunity ◽

Crispr Screen

Age-associated B cells (ABCs) accumulate after infection, during aging, and in systemic autoimmunity, contributing to pathogenesis in the latter1. To date, their precise ontogeny and whether they represent a B cell subset distinct from the other major effector subsets - plasma cells and memory B cells - remain unknown. We asked whether ABC formation requires the expression of a unique transcription factor. Here, we used single-cell RNA sequencing to identify differentially-expressed transcription factors shared by ABCs from both lupus patient and lupus-prone mice. Using an arrayed CRISPR screen, we show Zeb2 is essential for ABC differentiation in vitro. Selective deficiency of Zeb2 in B cells also leads to impaired ABC formation in vivo. Zeb2 drove ABC specification by directly binding the promoters of ABC signature genes including Itgax and Itgam encoding for CD11c and CD11b respectively. Zeb2 also targeted molecules involved in cytokine receptor signaling via the JAK/STAT pathway including Il21r and Socs5. Administration of the approved JAK/STAT inhibitor tofacitinib effectively ameliorated ABC accumulation in mice and patients with autoimmunity. This study reveals ABCs represent a distinct B cell subset dependent on Zeb2 expression, and provides a rationale for the use of JAK/STAT inhibitors to treat ABC-mediated autoimmune diseases.

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B cell–intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Blood ◽

10.1182/blood-2011-09-379412 ◽

2012 ◽

Vol 119 (12) ◽

pp. 2819-2828 ◽

Cited By ~ 62

Author(s):

Mike Recher ◽

Siobhan O. Burns ◽

Miguel A. de la Fuente ◽

Stefano Volpi ◽

Carin Dahlberg ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Plasma Cells ◽

Autoantibody Production ◽

Marginal Zone B Cells ◽

Wiskott Aldrich Syndrome ◽

Germinal Center B Cells

Abstract Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell–intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

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Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

Developmental Immunology ◽

10.1080/1044667031000088057 ◽

2002 ◽

Vol 9 (2) ◽

pp. 86-95 ◽

Cited By ~ 8

Author(s):

Denise A. Kaminski ◽

John J. Letterio ◽

Peter D. Burrows

Keyword(s):

B Cells ◽

Growth Factor ◽

B Cell ◽

Transforming Growth Factor ◽

Plasma Cells ◽

Population Analysis ◽

Cell Development ◽

B Cell Development

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

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

Cell Regulation ◽

10.1091/mbc.1.1.27 ◽

1989 ◽

Vol 1 (1) ◽

pp. 27-35 ◽

Cited By ~ 272

Author(s):

R D Sanderson ◽

P Lalor ◽

M Bernfield

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

B Lymphocyte ◽

Plasma Cells ◽

Receptor Expression ◽

Cell Stage ◽

Precursor B Cells

Lymphopoietic cells require interactions with bone marrow stroma for normal maturation and show changes in adhesion to matrix during their differentiation. Syndecan, a heparan sulfate-rich integral membrane proteoglycan, functions as a matrix receptor by binding cells to interstitial collagens, fibronectin, and thrombospondin. Therefore, we asked whether syndecan was present on the surface of lymphopoietic cells. In bone marrow, we find syndecan only on precursor B cells. Expression changes with pre-B cell maturation in the marrow and with B-lymphocyte differentiation to plasma cells in interstitial matrices. Syndecan on B cell precursors is more heterogeneous and slightly larger than on plasma cells. Syndecan 1) is lost immediately before maturation and release of B lymphocytes into the circulation, 2) is absent on circulating and peripheral B lymphocytes, and 3) is reexpressed upon their differentiation into immobilized plasma cells. Thus, syndecan is expressed only when and where B lymphocytes associate with extracellular matrix. These results indicate that B cells differentiating in vivo alter their matrix receptor expression and suggest a role for syndecan in B cell stage-specific adhesion.

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Specific In Vivo Deletion of B-Cell Subpopulations Expressing Human Immunoglobulins by the B-Cell Superantigen Protein L

Infection and Immunity ◽

10.1128/iai.72.6.3515-3523.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3515-3523 ◽

Cited By ~ 16

Author(s):

Muriel Viau ◽

Nancy S. Longo ◽

Peter E. Lipsky ◽

Lars Björck ◽

Moncef Zouali

Keyword(s):

B Cells ◽

B Cell ◽

Immune Defense ◽

Protein L ◽

Variable Regions ◽

Marginal Zone B Cells ◽

Human Immunoglobulins ◽

Cell Immunity ◽

B Cell Subsets

ABSTRACT Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.

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KLF4 suppresses transformation of pre-B cells by ABL oncogenes

Blood ◽

10.1182/blood-2006-03-011106 ◽

2006 ◽

Vol 109 (2) ◽

pp. 747-755 ◽

Cited By ~ 49

Author(s):

Michael G. Kharas ◽

Isharat Yusuf ◽

Vanessa M. Scarfone ◽

Vincent W. Yang ◽

Julia A. Segre ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

Tumor Suppressor ◽

B Cell ◽

Cell Activation ◽

Cell Transformation ◽

Cell Lineage ◽

Cell Types ◽

B Cell Malignancies

Abstract Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre–B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

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Cells Expressing the Epstein-Barr Virus Growth Program Are Present in and Restricted to the Naive B-Cell Subset of Healthy Tonsils

Journal of Virology ◽

10.1128/jvi.74.21.9964-9971.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 9964-9971 ◽

Cited By ~ 78

Author(s):

Alexandra M. Joseph ◽

Gregory J. Babcock ◽

David A. Thorley-Lawson

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Subset ◽

Pcr Analysis ◽

Virus Growth ◽

Barr Virus ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr

ABSTRACT In this paper we demonstrate, for the first time, that Epstein-Barr virus (EBV)-infected cells expressing the lymphoblastoid growth program are present in healthy carriers of the virus. Previously we observed that latently infected naive B cells are present in tonsils only when viral replication is detected, suggesting that these may represent newly infected B cells. We have tested this idea by performing a reverse transcription-PCR analysis for the expression of latent genes (EBNA2 and the EBNA3s) that are characteristically expressed only by newly infected cells expressing the growth latency program. EBNA2 expression is regularly detected in purified naive (IgD+) tonsillar B cells (13 of 16 tonsils tested) but was never found in the IgD− population (0 of 16). More detailed analysis revealed that the mRNAs for the latent genes EBNA1 (3 of 3 tonsils tested), EBNA3a (3 of 5), EBNA3b (3 of 5), EBNA3c (3 of 5), LMP1 (6 of 6), and LMP2 (5 of 6) were also present in the IgD+ population, but the EBNA1Q-K transcript, characteristic of nonlymphoblastoid forms of latency, was never detected (0 of 6). Finally, we demonstrate that the latently infected naive (IgD+) cells express CD80 (B7.1), a marker characteristically expressed on activated naive lymphoblasts but absent from resting naive B cells. The infected naive (IgD+) population in the tonsil therefore has the viral and cellular phenotype of a B-cell directly infected with EBV—an activated lymphoblast expressing the growth program.

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Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help

Journal of Experimental Medicine ◽

10.1084/jem.20060701 ◽

2006 ◽

Vol 203 (8) ◽

pp. 1985-1998 ◽

Cited By ~ 33

Author(s):

Laura Mandik-Nayak ◽

Jennifer Racz ◽

Barry P. Sleckman ◽

Paul M. Allen

Keyword(s):

Lymph Node ◽

T Cell ◽

B Cells ◽

B Cell ◽

Marginal Zone ◽

B Cell Tolerance ◽

Marginal Zone B Cells ◽

T Cell Help

In K/BxN mice, arthritis is induced by autoantibodies against glucose-6-phosphate-isomerase (GPI). To investigate B cell tolerance to GPI in nonautoimmune mice, we increased the GPI-reactive B cell frequency using a low affinity anti-GPI H chain transgene. Surprisingly, anti-GPI B cells were not tolerant to this ubiquitously expressed and circulating autoantigen. Instead, they were found in two functionally distinct compartments: an activated population in the splenic marginal zone (MZ) and an antigenically ignorant one in the recirculating follicular/lymph node (LN) pool. This difference in activation was due to increased autoantigen availability in the MZ. Importantly, the LN anti-GPI B cells remained functionally competent and could be induced to secrete autoantibodies in response to cognate T cell help in vitro and in vivo. Therefore, our study of low affinity autoreactive B cells reveals two distinct but potentially concurrent mechanisms for their activation, of which one is T cell dependent and the other is T cell independent.

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Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome

Blood ◽

10.1182/blood-2011-03-340711 ◽

2012 ◽

Vol 119 (19) ◽

pp. 4395-4407 ◽

Cited By ~ 33

Author(s):

Alexander Astrakhan ◽

Blythe D. Sather ◽

Byoung Y. Ryu ◽

Socheath Khim ◽

Swati Singh ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Types ◽

Gene Correction ◽

Wiskott Aldrich Syndrome ◽

Advantages And Disadvantages ◽

Life Threatening ◽

Lentiviral Gene Therapy ◽

High Level

AbstractThe immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)–deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.

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BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation

Blood ◽

10.1182/blood-2008-10-185421 ◽

2009 ◽

Vol 113 (17) ◽

pp. 3969-3977 ◽

Cited By ~ 102

Author(s):

Julia Eckl-Dorna ◽

Facundo D. Batista

Keyword(s):

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Formation ◽

Specific Class ◽

B Cell Differentiation ◽

Enhanced Production ◽

Stimulation Of

Abstract The activation of Toll-like receptor 9 (TLR9) expressed within B cells is associated with enhanced humoral immunity. However the role of TLR9 in the stimulation of B-cell responses, and more specifically in shaping the outcome of B-cell differentiation, remains unclear. Here, we observed that immunization with the TLR9 agonist CpG linked to protein antigen gave rise to enhanced production of antigen-specific class-switched antibodies in vivo. Unlike dendritic cells, B cells are unable to acquire these conjugates by macropinocytosis and instead depend on uptake through a signaling-competent B-cell receptor (BCR), provided the overall BCR-antigen avidity exceeds a defined threshold. The resultant stimulation of intrinsic TLR9 leads to enhanced antigen-specific B-cell proliferation and differentiation to form extrafollicular plasma cells. Thus, the direct conjugation of antigen and CpG reveals a mechanism that may operate during the initiation of primary immune responses, and may prove useful as a strategy for the design of adjuvants suitable for vaccinations.

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