An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Chicaybam, Leonardo; Sodré, Andressa L.; Curzio, Bianca Azevedo; Bonamino, Martin H.

doi:10.1371/journal.pone.0060298

Cited by 125 publications

(137 citation statements)

References 45 publications

(41 reference statements)

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“…Transfection of Human and Mouse B Cells with siRNA. B cells were transfected using electroporation following a protocol previously described for primary T cells (37). The 5-10 × 10 6 cells were resuspended in 100 μL electroporation buffer.…”

Section: Methodsmentioning

confidence: 99%

Nuclear TRAF3 is a negative regulator of CREB in B cells

Mambetsariev

Lin

Stunz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The adaptor protein TNF receptor-associated factor 3 (TRAF3) regulates signaling through B-lymphocyte receptors, including CD40, BAFF receptor, and Toll-like receptors, and also plays a critical role inhibiting B-cell homoeostatic survival. Consistent with these findings, loss-of-function human TRAF3 mutations are common in B-cell cancers, particularly multiple myeloma and B-cell lymphoma. B cells of B-cellspecific TRAF3−/− mice (B-Traf3) display remarkably enhanced survival compared with littermate control (WT) B cells. The mechanism for this abnormal homeostatic survival is poorly understood, a key knowledge gap in selecting optimal treatments for human B-cell cancers with TRAF3 deficiency. We show here for the first time to our knowledge that TRAF3 is a resident nuclear protein that associates with the transcriptional regulator cAMP response element binding protein (CREB) in both mouse and human B cells. The TRAF-C domain of TRAF3 was necessary and sufficient to localize TRAF3 to the nucleus via a functional nuclear localization signal. CREB protein was elevated in TRAF3 −/− B cells, without change in mRNA, but with a decrease in CREB ubiquitination. CREB-mediated transcriptional activity was increased in TRAF3-deficient B cells. Consistent with these findings, Mcl-1, an antiapoptotic target of CREBmediated transcription, was increased in the absence of TRAF3 and enhanced Mcl-1 was suppressed with CREB inhibition. TRAF3-deficient B cells were also preferentially sensitive to survival inhibition with pharmacologic CREB inhibitor. Our results identify a new mechanism by which nuclear TRAF3 regulates B-cell survival via inhibition of CREB stability, information highly relevant to the role of TRAF3 in B-cell malignancies.T RAF3 (TNF receptor-associated factor 3) is an important member of the TRAF adaptor family with distinct cell and context-specific roles (1). Our group previously generated CD19 Cre TRAF3 flox/flox mice (B-Traf3 −/− ) that lack TRAF3 specifically in B cells. This deletion results in remarkably enhanced B-cell survival in vitro and in vivo (2). Aged B-Traf3 −/− mice have substantially higher incidence of spontaneous B-cell lymphoma, further supporting the tumor-suppressive role of TRAF3 in B cells (3). Studies of human tumors identified loss-of-function TRAF3 mutations in nearly 20% of multiple myeloma (MM); TRAF3 is now recognized as one of the top 11 genes mutated in two-thirds of MM tumors (4). Additionally, ≥15% of diffuse large B-cell lymphomas are now known to harbor TRAF3 mutations (5, 6). Lesions in human TRAF3 genes are also seen in Hodgkin's lymphoma (7) and associated with particular chromosome 14 deletions in various B-cell lymphomas (8). Interestingly, Traf3 mutations are also common in canine B-cell lymphomas (5).TRAF3 is a negative regulator of the noncanonical NF-κB (NF-κB2) pathway, and enhanced survival in TRAF3-deficient B cells is associated with constitutive activation of NF-κB2 (2, 9). BAFF binds to BAFF receptor (BAFFR) to activate a complex signaling cascade that includes ...

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Section: Methodsmentioning

confidence: 99%

Nuclear TRAF3 is a negative regulator of CREB in B cells

Mambetsariev

Lin

Stunz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Electroporation was performed as previously described [13]. Briefly, 2 x 10 6 MDA-MB 231 cells were ressuspended in 100 μl of electroporation buffer (5 mM KCl; 15 mM MgCl 2 ; 120 mM Na 2 HPO 4 /NaH 2 PO 4 pH 7.2; 25 mM Sodium Succinate; 25 mM Manitol) containing 4 μg of the reporter system plasmid 7xTcf-FFluc//SV40-PuroR (7TFP, Addgene plasmid 24308) to evaluate the activation of Wnt signaling [14] and 0.4 μg of TKRenilla plasmid (Promega, USA).…”

Section: Cell Electroporation and Luciferase Assaymentioning

confidence: 99%

Membrane cholesterol depletion reduces breast tumor cell migration by a mechanism that involves non-canonical Wnt signaling and IL-10 secretion

et al. 2016

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Background: Migration and invasion are hallmarks of cancer cells that allow their dissemination to other tissues. Studying the cellular and molecular basis of cancer cell migration can help us to understand and control cancer metastasis. Many membrane molecules have been described as being involved in tumor cell migration, and among them is cholesterol. Method: In the present study we investigated the role of membrane cholesterol in the migration of breast tumor cells. The human mammary gland/breast epithelial cell line MDA-MB 231 was used and membrane cholesterol was depleted with methyl-β-cyclodextrin (MbCD). Cell migration was measured in a cell-based scratch assay and the involvement of the Wnt signaling was tested using Lef-1/TCF reporter activation in permanently transfected MDA-MB 231 cells. Cell morphology was analyzed using fluorescent phalloidin to label F-actin structures.

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“…The leukocytes were harvested of white blood cell reduction filters, and PBMCs were isolated by Ficoll density gradient centrifugation. hIRF2BP2-A overexpression in these cells was obtained by electroporation using AMAXA Nucleofactor IIb (Lonza, Basel Switzerland), according to a previously described protocol using the homemade buffer set 1SM [18]. Cells were stimulated with PMA (10 nM) and ionomycin (1 mM), 24 h after electroporation for an additional 24 h. Next, CD25 surface expression was analyzed by flow cytometry.…”

Section: Pbmc Transfection and Analysismentioning

confidence: 99%

IRF2BP2 transcriptional repressor restrains naive CD4 T cell activation and clonal expansion induced by TCR triggering

Sécca

Faget

Hanschke

et al. 2016

Journal of Leukocyte Biology

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CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iT but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli.

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An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Cited by 125 publications

References 45 publications

Nuclear TRAF3 is a negative regulator of CREB in B cells

Nuclear TRAF3 is a negative regulator of CREB in B cells

Membrane cholesterol depletion reduces breast tumor cell migration by a mechanism that involves non-canonical Wnt signaling and IL-10 secretion

IRF2BP2 transcriptional repressor restrains naive CD4 T cell activation and clonal expansion induced by TCR triggering

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