BAFF, IL‐4 and IL‐21 separably program germinal center‐like phenotype acquisition, BCL6 expression, proliferation and survival of CD40L‐activated B cells
            in vitro

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

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A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication

Blood ◽

10.1182/blood.v95.7.2321 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2321-2328 ◽

Cited By ~ 53

Author(s):

Kazuhiko Kuwahara ◽

Mikoto Yoshida ◽

Eisaku Kondo ◽

Atsuko Sakata ◽

Yuko Watanabe ◽

...

Keyword(s):

Dna Replication ◽

B Cells ◽

Germinal Center ◽

Messenger Rna ◽

Nuclear Protein ◽

Homologous Region ◽

Follicular Dendritic Cells ◽

Nuclear Phosphoprotein ◽

Deficient Mice

Abstract Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-μ and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.

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Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas

Blood ◽

10.1182/blood-2005-06-2342 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4345-4350 ◽

Cited By ~ 102

Author(s):

Dörte Bechtel ◽

Julia Kurth ◽

Claus Unkel ◽

Ralf Küppers

Keyword(s):

B Cells ◽

Cell Lines ◽

Germinal Center ◽

Somatic Hypermutation ◽

Cell Receptor ◽

Lymphoproliferative Disease ◽

Barr Virus ◽

Classic Hodgkin Lymphoma ◽

Epstein Barr

In classic Hodgkin lymphoma (HL) and posttransplantation lymphoproliferative disease (PTLD), 2 malignancies frequently associated with Epstein-Barr virus (EBV), the tumor cells often appear to derive from B-cell receptor (BCR)–deficient and therefore preapoptotic germinal center (GC) B cells. To test whether EBV can rescue BCR-less GC B cells, we infected human tonsillar CD77+ GC B cells in vitro with EBV. More than 60 monoclonal lymphoblastoid cell lines (LCLs) were established. Among these, 28 cell lines did not express surface immunoglobulin (sIg). Two of the sIg-negative cell lines carry obviously destructive mutations that have been introduced into originally functional VH gene rearrangements during the process of somatic hypermutation. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) showed that in most other lines the sIg deficiency was not simply the result of transcriptional down-regulation, but it was rather due to posttranscriptional defects. These findings strongly support the idea that EBV plays a central role in the pathogenesis of classic HL and PTLD by rescuing BCR-deficient, preapoptotic GC B cells from apoptosis, and that EBV infection renders the cells independent from survival signals normally supplied by a BCR. The monoclonal LCLs represent valuable models for early stages of lymphoma development in classic HL and PTLD.

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Inhibition of In Vitro Friend Murine Leukemia Virus Infection of Lipopolysaccharide-Activated B-Cells With Concanavalin A2

JNCI Journal of the National Cancer Institute ◽

10.1093/jnci/62.6.1497 ◽

1979 ◽

Keyword(s):

B Cells ◽

Virus Infection ◽

Concanavalin A ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Murine Leukemia ◽

Activated B Cells

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BCL-6 expression during B-cell activation

Blood ◽

10.1182/blood.v87.12.5257.bloodjournal87125257 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5257-5268 ◽

Cited By ~ 205

Author(s):

D Allman ◽

A Jain ◽

A Dent ◽

RR Maile ◽

T Selvaggi ◽

...

Keyword(s):

B Cells ◽

T Lymphocytes ◽

Germinal Center ◽

Cell Activation ◽

Interleukin 2 ◽

Large Cell ◽

Cd40 Ligand ◽

Mrna Levels ◽

Coding Region

Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5′ untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression.

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CD40 Ligand Activation Alters B Cell Migration to Secondary Lymphoid Organs.

Blood ◽

10.1182/blood.v108.11.935.935 ◽

2006 ◽

Vol 108 (11) ◽

pp. 935-935

Author(s):

Yvonne A. Efebera ◽

Tahamtan Ahmadi ◽

Amanda Flies ◽

David H. Sherr

Keyword(s):

B Cells ◽

Lymph Nodes ◽

B Cell ◽

Cd40 Ligand ◽

Lymphoid Organs ◽

Cell Populations ◽

Secondary Lymphoid Organs ◽

Activated B Cells

Abstract Background: An increased understanding of the requirements for antigen presentation has encouraged development of cell-based cancer vaccines. Trials using dendritic cells (DC) as antigen presenting cells (APC) for immunotherapy of several malignancies have shown considerable success. However, the difficulty in generating large numbers of DC required for these immunizations has led to the search for alternative APC. One such candidate is the CD40 ligand (CD40L)-activated B cell, populations of which can readily be expanded in vitro. To be an effective vehicle for antigen presentation to T cells, CD40L-activated B cells must be capable of migrating to secondary lymphoid organs. Therefore, CD40L-activated B cell migration following subcutaneous or intravenous injection was evaluated. Methods: Splenic B cells from GFP transgenic mice were activated with CD40L + IL-4 and expanded in vitro prior to i.v. or s.c. injection of 3–4 x 107 into C57BL/6 mice. Recipient mice were sacrificed 2 hrs or 1–14 days thereafter and the percentage of GFP+/B220+ B cells quantified in spleens and lymph nodes by flow cytometry. Localization of these cells within lymphoid organs was determined by immunohistochemistry. In some experiments, activated C57BL/6 B cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) to evaluate cell growth in vivo. Results: Murine B cell populations were readily expanded by culture on CD40L-transfected L cells in the presence of IL-4. CD40L-activated B cells expressed high levels of CD80, CD86, and LFA-1 but decreased levels of L-selectin relative to naive cells. Following i.v. injection, activated B cells were detected in spleens and lymph nodes within 1 day. Peak concentrations of activated B cells were noted in spleens and lymph nodes on days 7 (4.8% of injected cells) and 10 (1.25% of injected cells) respectively, suggesting expansion of the activated B cell population in vivo. Naive B cells injected i.v. were detected within 1 day but their number declined precipitously thereafter. Following s.c. injection, peak levels of CD40L-activated B cells were noted on day 5 (spleens) and day 7 (lymph nodes). As determined by immunohistochemistry, both CD40L-activated and naïve B cells injected i.v. appeared in B cell regions of spleens and lymph nodes. While the kinetics of accumulation of CD40L-activated B cells injected s.c. or i.v. were similar, s.c. injected CD40L-activated B cells homed to the T cell regions of spleens and lymph nodes. CFSE experiments indicated that these activated B cells continue to grow in vivo. In contrast, naïve B cells injected s.c. only appeared in B cell regions. Conclusion: CD40L-activated B cell populations can readily be expanded in vitro, CD40L-activated B cells migrate to secondary lymphoid organs even when injected s.c., activated B cell populations expand in vivo, and s.c. injected, CD40L-activated B cells preferentially home to T cell regions of secondary lymphoid organs. These results suggest that this effective APC may serve as an important vehicle for delivery and presentation of exogenous (e.g. tumor) antigens to T cells in vivo.

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