Functional analysis of the tumour suppressor gene PTEN in murine B cells and keratinocytes

2004 ◽  
Vol 32 (2) ◽  
pp. 362-365 ◽  
Author(s):  
A. Suzuki ◽  
T. Sasaki ◽  
T.W. Mak ◽  
T. Nakano
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Mutant Mice ◽  
Specific Gene ◽  
Marginal Zone B Cells ◽  
Specific Mutation ◽  
Phosphoinositide 3 Kinase

To investigate the roles of the PTEN (phosphatase and tensin homologue deleted from chromosome 10)/PI3K (phosphoinositide 3-kinase) signalling pathway in vivo, we have generated a series of mutant mice with null or tissue-specific gene-targeted deletions of Pten. Here we present our investigations of Pten function in B cells and keratinocytes in mice. Mice with a B cell-specific mutation of Pten showed increased serum autoantibodies and elevated numbers of B1a cells. Among conventional B (B2) cells in mutant spleens, numbers of marginal zone B cells were significantly increased, while those of follicular B cells were reciprocally decreased. Immunoglobulin class switch recombination was defective and associated with impaired induction of activation-induced cytidine deaminase. Mice with a keratinocyte-specific mutation of Pten exhibited epidermal hyperplasia, hyperkeratosis and accelerated skin morphogenesis. Within 3 weeks of birth, 90% of these animals died of malnutrition, possibly caused by hyperkeratosis of the oesophageal epithelia. Surviving mutant mice developed spontaneous skin tumours within 8.5 months of birth, and chemical treatment accelerated the onset of tumours. Our data show that PTEN is an important regulator in B cells and keratinocytes.

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

2004 ◽  
Vol 72 (6) ◽  
pp. 3515-3523 ◽  
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.

scholarly journals Sentinel interaction mapping – a generic approach for the functional analysis of human disease gene variants using yeast

2020 ◽  
Vol 13 (7) ◽  
pp. dmm044560
Author(s):  
Barry P. Young ◽  
Kathryn L. Post ◽  
Jesse T. Chao ◽  
Fabian Meili ◽  
Kurt Haas ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Human Disease ◽  
Disease Gene ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Disease Genes ◽  
Gene Variants ◽  
Human Disease Gene ◽  
Simple Growth

ABSTRACTAdvances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, ‘sentinel’ yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3251-3251
Author(s):  
Rita Shaknovich ◽  
Katia Basso ◽  
Govind Bhagat ◽  
Bachir Alobeid ◽  
Giorgio Cattoretti
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Subset ◽  
Cellular Transformation ◽  
Cell Types ◽  
Marginal Zone B Cells ◽  
Latently Infected ◽  
Protein Signature

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.

Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2397-2397
Author(s):  
Gabriel Brisou ◽  
Laurent Jallades ◽  
Alexandra Traverse-Glehen ◽  
Francoise Berger ◽  
Aurélie Verney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cytidine Deaminase ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Marginal Zone B Cells ◽  
Activation Induced Cytidine Deaminase ◽  
Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

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

2006 ◽  
Vol 203 (8) ◽  
pp. 1985-1998 ◽  
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.

Human splenic marginal zone B cells lack expression of activation-induced cytidine deaminase

2005 ◽  
Vol 35 (10) ◽  
pp. 3002-3007 ◽  
Author(s):  
Klaus Willenbrock ◽  
Berit Jungnickel ◽  
Martin-Leo Hansmann ◽  
Ralf Küppers
Keyword(s):  
B Cells ◽  
Marginal Zone ◽  
Cytidine Deaminase ◽  
Marginal Zone B Cells ◽  
Activation Induced Cytidine Deaminase

scholarly journals Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors

2021 ◽  
Author(s):  
Qi Zhang ◽  
Bingyan Wu ◽  
Qitong Weng ◽  
Fangxiao Hu ◽  
Yunqing Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Immune Memory ◽  
B Lymphopoiesis ◽  
Marginal Zone B Cells ◽  
Humoral Immune

AbstractRegeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

scholarly journals Sentinel Interaction Mapping (SIM) – A generic approach for the functional analysis of human disease gene variants using yeast

2020 ◽  
Author(s):  
Barry P Young ◽  
Kathryn L Post ◽  
Jesse T Chao ◽  
Fabian Meili ◽  
Kurt Haas ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Human Disease ◽  
Disease Gene ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Disease Genes ◽  
Gene Variants ◽  
Human Disease Gene ◽  
Simple Growth

AbstractAdvances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using Synthetic Dosage Lethality screening, “sentinel” yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in high-throughput fashion through simple growth assays using either solid or liquid media. Sentinel Interaction Mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

CLL Tumours with a Deletion of 11q Form Two Functional Subgroups Based on the Mutation Status of the Remaining ATM Allele.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 710-710
Author(s):  
Belinda Austen ◽  
Maria Podinovskaia ◽  
Claire Almond ◽  
Graham Fews ◽  
Anne Gardiner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Fish Analysis ◽  
Wild Type ◽  
Atm Gene ◽  
Atm Protein ◽  
11Q Deletion

Abstract Deletions in chromosome 11q are an established prognostic marker in CLL. One copy of the ATM gene is deleted in these tumours, as detected by FISH analysis. However, it remains unclear whether the ATM gene is the main tumour suppressor gene that is accounting for the poor outcome in tumours with 11q deletions. We have recently reported that patients whose tumours have mutations in the ATM gene have an impaired overall and treatment free survival. In our large cohort of 155 patients, tumours with an ATM mutation only partly correlated with tumours with an 11q deletion. We have therefore investigated the relationship between 11q deletions and mutations in the ATM gene. Using the highly sensitive DHPLC method, we have screened the 60 ATM coding exons for mutations in a cohort of 46 tumours, all with a deletion of chromosome 11q. We have found ATM mutations in 19 tumours, indicating a prevalence of 41%. The ATM protein is vital in the cell’s response to DNA damage including that induced by chemotherapy. ATM acts upstream from p53 and defects in ATM function, like p53, lead to impaired DNA damage induced apoptosis. Furthermore, we have previously shown that loss of ATM function is associated with both in vitro and in vivo chemo-resistance. Therefore, we next assessed whether the status of the remaining ATM allele in the 11q deleted tumours affected the response to DNA damage. Firstly we induced DNA damage with irradiation and measured both the phosphorylation of ATM protein targets and the induction of p53 dependent transcription responses in representative samples. We found that 11q deleted tumours with a remaining wild type ATM allele had responses that were similar to those seen in tumours with two wild type ATM alleles. In contrast, 11q deleted tumours with a mutation in the remaining ATM allele had defective DNA damage induced responses. We then analysed the effects of in vitro treatment with Fludarabine. First we demonstrated that fludarabine induces ATM dependent phophosphorylation responses in CLL tumours. Then we analysed its effect in the two 11q deleted CLL subgroups. We showed defective phosphorylation responses to fludarabine in the tumours with a mutation in the second ATM allele, but normal responses in those with a second wild type ATM allele. In summary, we have shown that approximately 40% of CLL tumours with an 11q deletion have a mutation in their remaining ATM allele. Furthermore, we have demonstrated that the 11q deleted tumours appear to form two functional subgroups based on the presence of a mutation in the remaining ATM allele. In contrast to the subgroup with a wild type ATM allele, CLL tumours with a mutant ATM allele have defective in vitro responses to DNA damage with both irradiation and fludarabine. We expect that the functional differences between the two 11q deleted subsets will translate into differences in clinical outcome.

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