Notch Regulation by the Fbw7/hcdc4/Sel-10 Ubiquitin Ligase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1420-1420
Author(s):  
Jonathan E. Grim ◽  
Olga Sala ◽  
Nack Gyun Chung ◽  
Jerald Radich ◽  
Barbara J. Varnum-Finney ◽  
...  
Keyword(s):  
T Cell ◽  
Ubiquitin Ligase ◽  
Heterozygous Mutation ◽  
Candidate Peptide ◽  
Human T Cell ◽  
Notch Protein ◽  
Important Regulator

Abstract T-cell neoplasms frequently sustain mutations in the Notch1 gene, leading to the expression of constitutively active Notch proteins. Such mutations often target the C-terminal PEST domain, which is known to be involved in protein stability. The ubiquitin ligase Fbw7/hdcd4/Sel-10 is a tumor suppressor that negatively regulates Notch function by targeting the Notch protein for ubiquitination and proteasomal degradation. Although the PEST domain is known to be important for Fbw7/Notch interactions, the specific residues that regulate binding of Notch to Fbw7 have not been defined. Based on the structural motifs (known as phosphodegrons) common to known substrates of Fbw7, we have identified two candidate peptide sequences within the Notch protein and have generated a series of mutants in these regions. Using co-immunoprecipitation assays, we show that one potential phosphodegron that is outside of the PEST domain does not appear to influence Notch binding to Fbw7. However, a second potential phosphodegron is present within the PEST domain and contains a conserved threonine residue (T2512) which is central to binding of Fbw7 to Notch. A mutant in which this residue is replaced by alanine (T2512A) shows a prolonged half life when compared to wild type Notch ICD, supporting its role in Notch stability. To evaluate the role of Fbw7 mediated Notch degradation in vitro and in vivo, we used lentiviral vectors to transfect hematopoietic cells with shRNA targeting Fbw7. These studies demonstrate that Fbw7 knockdown leads to phenotypes consistent with increased Notch activity. Because Notch is commonly mutated in human leukemias, we hypothesized that Fbw7 may also sustain mutations that lead to loss of Notch regulation. We evaluated primary human T cell leukemias for mutations in Fbw7 and found that 1 of 23 samples contains a heterozygous mutation in the Fbw7 common region (R505C). We show that this mutant is deficient in binding to Notch, suggesting that Fbw7 mutation may contribute to the deregulation of Notch that is commonly seen in T-cell neoplasms. Together, this work shows that Fbw7 is an important regulator of Notch function whose mutation may be an important step in leukemogenesis.

scholarly journals CCDC88B is a novel regulator of maturation and effector functions of T cells during pathological inflammation

2014 ◽  
Vol 211 (13) ◽  
pp. 2519-2535 ◽  
Author(s):  
James M. Kennedy ◽  
Nassima Fodil ◽  
Sabrina Torre ◽  
Silayuv E. Bongfen ◽  
Jean-Frédéric Olivier ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Coiled Coil ◽  
Cell Receptor ◽  
Pleiotropic Effect ◽  
Experimental Cerebral Malaria ◽  
A Genome ◽  
Important Regulator

We used a genome-wide screen in mutagenized mice to identify genes which inactivation protects against lethal neuroinflammation during experimental cerebral malaria (ECM). We identified an ECM-protective mutation in coiled-coil domain containing protein 88b (Ccdc88b), a poorly annotated gene that is found expressed specifically in spleen, bone marrow, lymph nodes, and thymus. The CCDC88B protein is abundantly expressed in immune cells, including both CD4+ and CD8+ T lymphocytes, and in myeloid cells, and loss of CCDC88B protein expression has pleiotropic effects on T lymphocyte functions, including impaired maturation in vivo, significantly reduced activation, reduced cell division as well as impaired cytokine production (IFN-γ and TNF) in response to T cell receptor engagement, or to nonspecific stimuli in vitro, and during the course of P. berghei infection in vivo. This identifies CCDC88B as a novel and important regulator of T cell function. The human CCDC88B gene maps to the 11q13 locus that is associated with susceptibility to several inflammatory and auto-immune disorders. Our findings strongly suggest that CCDC88B is the morbid gene underlying the pleiotropic effect of the 11q13 locus on inflammation.

scholarly journals Tax and Overlapping Rex Sequences Do Not Confer the Distinct Transformation Tropisms of Human T-Cell Leukemia Virus Types 1 and 2

2003 ◽  
Vol 77 (14) ◽  
pp. 7728-7735 ◽  
Author(s):  
Jianxin Ye ◽  
Li Xie ◽  
Patrick L. Green
Keyword(s):  
T Cells ◽  
T Cell ◽  
Leukemia Virus ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are distinct oncogenic retroviruses that infect several cell types but display their biological and pathogenic activity only in T cells. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4+ T cells, whereas HTLV-2 in vivo tropism is less clear but appears to favor CD8+ T cells. Both CD4+ and CD8+ T cells are susceptible to HTLV-1 and HTLV-2 infection in vitro, and HTLV-1 has a preferential immortalization and transformation tropism of CD4+ T cells, whereas HTLV-2 immortalizes and transforms primarily CD8+ T cells. The molecular mechanism that determines this tropism of HTLV-1 and HTLV-2 has not been determined. HTLV-1 and HTLV-2 carry the tax and rex transregulatory genes in separate but partially overlapping reading frames. Since Tax has been shown to be critical for cellular transformation in vitro and interacts with numerous cellular processes, we hypothesized that the viral determinant of transformation tropism is encoded by tax. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo), we constructed recombinants in which tax and overlapping rex genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex but with significantly altered activity compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells. Both recombinants were competent to transform T lymphocytes with an efficiency similar to that of the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4+ T cells and that HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8+ T cells. Our results indicate that tax/rex in different genetic backgrounds display altered functional activity but ultimately do not contribute to the different in vitro transformation tropisms. This first study with recombinants between HTLV-1 and HTLV-2 is the initial step in elucidating the different pathobiologies of HTLV-1 and HTLV-2.

On the role of APC-activation for in vitro versus in vivo T cell priming

2003 ◽  
Vol 225 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Tazio Storni ◽  
Martin F. Bachmann
Keyword(s):  
T Cell ◽  
T Cell Priming

Modeling Human T Cell Acute Lymphoblastic Leukemia In Vitro and In Vivo with LMO2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3648-3648
Author(s):  
James A Kennedy ◽  
Sara Berthiaume ◽  
Frederic Barabe
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cell ◽  
Leukemic Cell ◽  
Human Cells ◽  
Immunodeficient Mice ◽  
Human T Cell

Abstract Abstract 3648 The studies identifying gene translocations and mutations in T-ALL cell lines and/or in patients have contributed significantly to the understanding of the genetic abnormalities involved in T-ALL. However, studies on the biology of these genes, the targeted cells, the sequence and the number of hits required to convert a primary human hematopoietic stem cell (HSC)/progenitor cell into a fully transformed leukemic cell require good experimental models of human T cell development both in vivo and in vitro. The only in vivo model of human T cell leukemogenesis came unexpectedly from the gene therapy trial on patients with X-linked severe combined immunodeficiency (SCID-X1). Three to five years after gene therapy, 4 out of 10 patients in the trial developed clonal T-ALL. In these patients, retroviral integrations were found in proximity to the LMO2 promoter in the malignant clones, leading to aberrant expression of the oncogene. However, little is known on the effect of LMO2 overexpression in human cells and how it facilitates the development of T-ALL. We have developed in vivo and in vitro models to study the role of T cell oncogenes in human cells. Using the OP9-DL1 co-culture system to differentiate human HSC into mature T cells in vitro, we culture human HSC transduced with lentiviruses expressing LMO2. LMO2 overexpressing cells are blocked at the double negative stage (CD4-CD8-) of differentiation when co-cultured on OP9-Delta-Like1 stroma and proliferate 50 to 100 times more than control cells. However, these cells are not immortalized and cultures lasted approximately 80 days. LMO2 overexpression have no effect on myeloid differentiation in vitro. In vivo, LMO2 transduced human HSC/progenitor cells engraft the bone marrow of immunodeficient mice to levels comparable to control cells, while normal myeloid and B cell populations 20–24 weeks post-transplantation. LMO2 transduced cells have an increased capacity to generate T cells in the thymus in comparison to control cells (42% engraftment vs 8%, p<0.0001). Surprisingly, thymic and peripheral LMO2 cells are not blocked in their differentiation. LMO2 cells did not engraft secondary mice, confirming that LMO2 doesn't induce self-renewal of human HSC. However, the increase in thymic repopulation by LMO2 cells and the lack of differentiation block in vivo suggest that LMO2 overexpression generates an abnormal T cell population with an increase repopulation advantage (increase proliferation or decrease apoptosis) in the thymus which becomes the substrate for additional genetic/epigenetic events. To test this hypothesis, we tried to immortalize LMO2 cells in vitro with secondary hits. Our preliminary results show that insertional mutagenesis can immortalized LMO2 cells in vitro. However these cells are not able to engraft immunodeficient mice or generate leukemia in vivo. The addition of intracellular NOTCH to one immortalized LMO2 cell line allows these cells to engraft and generate human T-ALL in vivo. Globally, these results show that T cell oncogenes can be studied in primary human hematopoietic cells both in vitro and in vivo. Also, at least three hits are required to transform a human primary HSC/progenitor cell into a leukemic cell able to engraft and generate leukemia in vivo. It also suggests that a non-engrafting cell can be turned into a leukemic cell generating leukemia in vivo, implying that a cell can regain self-renewing properties. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Studying the immunosuppressive role of indoleamine 2,3-dioxygenase: tryptophan metabolites suppress rat allogeneic T-cell responses in vitro and in vivo

2005 ◽  
Vol 18 (1) ◽  
pp. 95-100 ◽  
Author(s):  
Thomas M. Bauer ◽  
Lucian P. Jiga ◽  
Jing-Jing Chuang ◽  
Marco Randazzo ◽  
Gerhard Opelz ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Cell Responses ◽  
Indoleamine 2,3 Dioxygenase ◽  
Tryptophan Metabolites

scholarly journals IL-27 promotes T cell–dependent colitis through multiple mechanisms

2010 ◽  
Vol 208 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Jennifer H. Cox ◽  
Noelyn M. Kljavin ◽  
Nandhini Ramamoorthi ◽  
Lauri Diehl ◽  
Marcel Batten ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Intestinal Inflammation ◽  
Interferon Γ ◽  
Transfer Model ◽  
Proinflammatory Role ◽  
Immune Pathology

Interleukin-27 (IL-27) is a cytokine known to have both proinflammatory and immunoregulatory functions. The latter appear to dominate in vivo, where IL-27 suppresses TH17 responses and promotes the differentiation of Tr1 cells expressing interferon-γ and IL-10 and lacking forkhead box P3 (Foxp3). Accordingly, IL-27 receptor α (Il27ra)–deficient mice suffer from exacerbated immune pathology when infected with various parasites or challenged with autoantigens. Because the role of IL-27 in human and experimental mouse colitis is controversial, we studied the consequences of Il27ra deletion in the mouse T cell transfer model of colitis and unexpectedly discovered a proinflammatory role of IL-27. Absence of Il27ra on transferred T cells resulted in diminished weight loss and reduced colonic inflammation. A greater fraction of transferred T cells assumed a Foxp3+ phenotype in the absence of Il27ra, suggesting that IL-27 functions to restrain regulatory T cell (Treg) development. Indeed, IL-27 suppressed Foxp3 induction in vitro and in an ovalbumin-dependent tolerization model in vivo. Furthermore, effector cell proliferation and IFN-γ production were reduced in the absence of Il27ra. Collectively, we describe a proinflammatory role of IL-27 in T cell–dependent intestinal inflammation and provide a rationale for targeting this cytokine in pathological situations that result from a breakdown in peripheral immune tolerance.

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