Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Mol Biotechnol ; 47(3): 262-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20938757

ABSTRACT

Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14+ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80-90 and 70-90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.


Subject(s)
Dendritic Cells/metabolism , Genetic Vectors/genetics , Lentivirus/genetics , Cells, Cultured , Flow Cytometry , Humans , Phagocytosis/genetics , Phagocytosis/physiology , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Transduction, Genetic
2.
Mol Immunol ; 47(6): 1255-61, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20071030

ABSTRACT

Interleukin (IL)-23 is a heterodimeric cytokine composed of the IL-23-specific subunit p19 and the p40 subunit which also constitutes part of IL-12. IL-23 propagates development of Th17 cells, a novel T cell subset which produces IL-17 but no interferon-gamma or IL-4. For both, IL-23 and IL-23-driven IL-17, a crucial role in autoimmune diseases such as experimental autoimmune encephalomyelitis, collagen-induced arthritis, and colitis is well accepted. Recent studies indicate that there is also a role for IL-23 and IL-17 in tumorigenesis, promoting tumor growth and vascularization, and affecting tumor incidence. We show that human CD14(+) peripheral blood monocyte-derived dendritic cells (DC), as used for clinical applications in anti-tumor immunization strategies, produce high amounts of IL-23. CD40-triggering of immature and mature DC but not of primary monocytes induced a rapid expression of high levels of IL-23, free p40, and minor levels of IL-12. Upon stimulation of DC subsets with a variety of different danger signals such as single stranded and double stranded RNA, bacterial components or viral infections, IL-23 expression pattern was analyzed. Interestingly, co-stimulation with CD40L enabled IL-23 expression by DC subsets towards danger signals to which they have been unresponsive upon single stimulation. Furthermore, we detected two novel splice variants of the IL-23-specific subunit p19 that could be associated with the regulation of IL-23 expression. Data presented here might have an impact on DC-based cancer vaccination strategies and contribute to a better understanding of the complex regulation of the heterodimeric cytokine IL-23.


Subject(s)
CD40 Ligand/immunology , Dendritic Cells/immunology , Interleukin-23/immunology , Signal Transduction/immunology , Alternative Splicing/genetics , Amino Acid Sequence , Base Sequence , Cell Differentiation , Dendritic Cells/cytology , Dendritic Cells/microbiology , Dendritic Cells/virology , Humans , Interleukin-12 Subunit p40/metabolism , Interleukin-23 Subunit p19/chemistry , Interleukin-23 Subunit p19/genetics , Molecular Sequence Data , Receptors, Pattern Recognition/immunology
3.
J Allergy Clin Immunol ; 122(5): 890-2, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18805577

ABSTRACT

In March 2006, 6 healthy volunteers experienced serious adverse reactions during a first-in-human clinical trial of the superagonistic anti-CD28 mAb TGN1412. A first investigation excluded contaminations of the drug product or protocol irregularities as the root cause. Later, an expert scientific group convened in the United Kingdom to develop recommendations pertinent to minimizing risks of first-in-human clinical trials. The expert scientific group concluded from in silico calculations that at the initial dose of 0.1 mg/kg, which was adjusted on the basis of the no observed adverse effect level, approximately 86.2% to 90.9% CD28 receptor occupancy was obtained. Here we developed a flow cytometric method that revealed receptor occupancy of approximately 45% to 80% under the above conditions. Thus we present a method to experimentally determine receptor occupancy that can be taken as one parameter to define the minimal anticipated biological effect level as the basis for calculating safer starting doses for first-in-human clinical trials for products in which a potential risk has been identified. Additional measures are being discussed that will help to significantly improve safety of first-in-human clinical trials.


Subject(s)
Antibodies, Monoclonal/adverse effects , Receptors, Immunologic/immunology , Toxicity Tests , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized , CD28 Antigens/immunology , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Flow Cytometry , Humans , No-Observed-Adverse-Effect Level
4.
PLoS One ; 3(3): e1708, 2008 Mar 05.
Article in English | MEDLINE | ID: mdl-18320029

ABSTRACT

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4+ T cells but not in cynomolgus T lymphocytes. The sustained Ca++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-gamma and TNF-alpha. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-gamma and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.


Subject(s)
Antibodies, Monoclonal/pharmacology , CD28 Antigens/immunology , Calcium/metabolism , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Blotting, Western , Cell Proliferation , Flow Cytometry , Humans , Interferon-gamma/metabolism , Interleukin-2/metabolism , Lymphocyte Activation , Macaca fascicularis , Macaca mulatta , Quinuclidines/immunology , Quinuclidines/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Tumor Necrosis Factor-alpha/metabolism , src-Family Kinases/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...