Generation of T-cell receptor retrogenic mice

Holst, Jeff; Szymczak-Workman, Andrea L.; Vignali, Kate M.; Burton, Amanda R.; Workman, Creg J.

doi:10.1038/nprot.2006.61

Cited by 235 publications

(311 citation statements)

References 20 publications

(32 reference statements)

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“…Encouraged by the successful use of 2A peptides in g-retroviral vectors to express multiple genes in one construct, 24,25,27,51 we attempted to develop lentiviral vectors expressing antitumor antigen TCR using different 2A peptides for adoptive immunotherapy. The main advantage of using 2A peptides in the construction of bicistronic vectors is the potential for co-expression of both genes at equal levels.…”

Section: Discussionmentioning

confidence: 99%

“…Both T-cell subsets exhibited significant release of IFN-g to HLA A2-matched tumor cells (Figure 6c). Lastly, tumor cell lysis by the TCR-engineered CD4/CD8 lymphocytes was determined by 51 Cr release assay following co-culture with two HLA A2 + and two HLA A2 À melanoma lines (Figure 6d). Both CD4 and CD8 TCR-transduced T cells demonstrated specific lytic activity and as expected, CD8T cells lysed tumors to a greater extent than CD4T cells.…”

Section: One Furin Site Is Sufficient For Tcr Maturationmentioning

confidence: 99%

“…Harvested supernatants were counted using a Wallac 1470 Wizard automatic gamma counter. Total and spontaneous 51 Cr release was determined by incubating 2 Â 10 3 labeled target cells in either 2% SDS or medium alone for above condition, respectively. Each data point was conducted as a mean of quadruplicate wells.…”

Section: Cr Release Assaymentioning

confidence: 99%

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Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

et al. 2008

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In human gene therapy applications, lentiviral vectors may have advantages over g-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike g-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust antimelanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

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Section: Discussionmentioning

confidence: 99%

Section: One Furin Site Is Sufficient For Tcr Maturationmentioning

confidence: 99%

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Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

et al. 2008

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“…Murine stem cell virus-based retroviral expression vectors encoding the DQ8/Ins complexes were constructed using standard molecular biology procedures. The constructs encode the complexes as a single ORF, comprising the DQ8 α-and β-chains separated by a picornovirus 2A peptide (47), and an internal ribosome entry site-driven GFP. The insulin peptide is fused via a flexible linker to the N terminus of the mature β-chain (48) (Fig.…”

Section: Methodsmentioning

confidence: 99%

Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

Yang¹,

Chow²,

Sosinowski

et al. 2014

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

106

149

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Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA g7 in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a lowaffinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the lowaffinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.antigen presentation | self-antigen | MHCII tetramers T ype 1 diabetes (T1D) is a polygenic T-cell-mediated autoimmune disease with strong genetic linkages to the MHC class II and insulin promoter regions (1). Class II molecules are fundamental for CD4 + T-cell activation, whereas the insulin promoter polymorphism can modulate insulin levels in the thymus thereby influencing the threshold of selection for insulinspecific autoreactive T cells (2, 3). In the nonobese diabetic (NOD) mouse model of autoimmune mediated diabetes, insulinspecific IA g7 -restricted CD4 + T cells have been strongly implicated in β-cell destruction. In prediabetic NOD mice, 50% of the T-cell clones established from islet-infiltrating lymphocytes were insulin-specific and the majority of these clones recognized the insulin B:9-23 (B:9-23 SHLVEALYLVCGERG) epitope (4, 5). Moreover, substitution of a single residue in the B:9-23 region abrogated development of diabetes in a transgenic mouse model (6, 7). Thus, in the murine NOD model, the IA g7 -restricted B:9-23 epitope is considered to be pivotal in the development of diabetes.The position or "register" that B:9-23 occupies within the IA g7 binding groove has been a controversial but important question. At least three binding registers (R) have been considered, defined here by which B:9-23 amino acid occupies the first binding pocket (p1) position in the IA g7 ...

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“…Retroviral producer cell lines were generated as described (28,31). The protocol for the retroviralmediated stem cell gene transfer has also been described (32).…”

Section: Methodsmentioning

confidence: 99%

A trans -Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo

Lieu

Lock

Hammond

et al. 2008

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

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The transmembrane precursor of tumor necrosis factor-␣ (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNAvector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery. intracellular trafficking ͉ RNAi transgenic mice ͉ TNF secretion ͉ inflammation ͉ membrane transport T umor necrosis factor-␣ (TNF) is the main proinflammatory cytokine made and secreted by inflammatory macrophages. Early release of TNF in response to LPS or other inflammatory signals enhances the activation and recruitment of T cells and ensures robust innate and acquired immune responses (1). The excessive secretion of TNF is also a prevalent and clinically significant problem in acute inflammation and in chronic inflammatory disease (2). Anti-TNF treatments have shown success in the treatment of rheumatoid arthritis, inflammatory bowel disease, and other conditions (3). Now improved antiTNF strategies that may offer more constrained or cell type specific control of TNF secretion are being sought. This requires identification of molecular mediators of TNF secretion, particularly those that can be targeted to block TNF release.We have characterized the secretory pathway for TNF in activated macrophages and identified key organelles and components of the trafficking machinery whose expression or function is up-regulated by LPS to support cytokine secretion (4-6). Among these are members of the SNARE family of membrane fusion proteins required at different points along this ...

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Generation of T-cell receptor retrogenic mice

Cited by 235 publications

References 20 publications

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

A trans -Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo

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