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1.
Nat Immunol ; 14(8): 858-66, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23793062

ABSTRACT

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.


Subject(s)
CARD Signaling Adaptor Proteins/immunology , CD28 Antigens/immunology , Carrier Proteins/immunology , Guanylate Cyclase/immunology , Protein Kinase C/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Regulatory/immunology , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/genetics , Flow Cytometry , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins , Microscopy, Confocal , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Alignment , Sequence Analysis, DNA , Specific Pathogen-Free Organisms
2.
Ther Deliv ; 4(6): 673-85, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23738666

ABSTRACT

BACKGROUND: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.


Subject(s)
Hydrogels , Tissue Scaffolds , Transfection/methods , Animals , Gene Silencing , Humans , Hydrogels/chemistry , Microscopy, Fluorescence , Transgenes
3.
Eur J Immunol ; 42(9): 2395-408, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22684987

ABSTRACT

Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.


Subject(s)
DNA-Binding Proteins/genetics , Neutropenia/genetics , Point Mutation , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , Antigens, Ly/genetics , Antigens, Ly/metabolism , Arthritis/genetics , Arthritis/metabolism , B-Lymphocytes/metabolism , Bone Marrow/metabolism , CD11b Antigen/genetics , CD11b Antigen/metabolism , DNA-Binding Proteins/metabolism , Ethylnitrosourea , Female , Inflammation/genetics , Inflammation/metabolism , Lymphocytes/metabolism , Lymphopoiesis/genetics , Male , Mice , Mice, Inbred C57BL , Neutropenia/chemically induced , Neutrophils/metabolism , Repressor Proteins/metabolism , T-Lymphocytes/metabolism , Transcription Factors/metabolism , Transcription, Genetic
4.
Science ; 335(6066): 344-8, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22267813

ABSTRACT

Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.


Subject(s)
Antigens, Ly/physiology , DNA-Binding Proteins/genetics , Herpesviridae Infections/immunology , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 1/physiology , T-Lymphocytes/immunology , Transcription Factors/genetics , Adaptive Immunity , Amino Acid Substitution , Animals , Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Antigens, Ly/genetics , Antigens, Ly/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , DNA-Binding Proteins/physiology , Down-Regulation , Genetic Complementation Test , Herpesviridae Infections/virology , Immunologic Memory , Listeriosis/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muromegalovirus/physiology , Mutagenesis , Natural Cytotoxicity Triggering Receptor 1/antagonists & inhibitors , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/immunology , Transcription Factors/physiology , Transcription, Genetic , Viral Load
5.
Biotechniques ; 50(3): 187-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21486240

ABSTRACT

Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.


Subject(s)
DNA/administration & dosage , Magnetics , Neural Stem Cells/cytology , Transfection/methods , Animals , Cells, Cultured , Mice , Neurogenesis , Transfection/economics
6.
Ther Deliv ; 2(4): 471-82, 2011 Apr.
Article in English | MEDLINE | ID: mdl-22826855

ABSTRACT

In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either 'naked' or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.


Subject(s)
Biomedical Technology/methods , Gene Transfer Techniques , Genetic Therapy/methods , Magnetic Fields , Metal Nanoparticles/therapeutic use , Nucleic Acids/administration & dosage , Animals , Genetic Vectors , Humans , Models, Biological , Transfection/methods
7.
Immunity ; 31(2): 197-208, 2009 Aug 21.
Article in English | MEDLINE | ID: mdl-19682930

ABSTRACT

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class II/immunology , Lymphoproliferative Disorders/immunology , Membrane Proteins/immunology , Phosphoproteins/immunology , Receptors, Antigen, T-Cell/immunology , Adaptor Proteins, Signal Transducing/genetics , Adoptive Transfer , Animals , CD28 Antigens/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cytokines/immunology , Cytokines/metabolism , Histocompatibility Antigens Class II/metabolism , Lymphoproliferative Disorders/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mutation , Phosphoproteins/genetics , Phosphorylation/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology
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