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Wolfgang Uckert

Publications and source records attributed to Wolfgang Uckert.

12 recordsLinked to original sources
PubMed2006

Designer T cells by T cell receptor replacement.

T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong--in terms of cell surface expression--and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRalpha/beta combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering.

Animals↗
PubMed2005

Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells.

Cytotoxic T lymphocytes (CTLs) specific for an HLA-A2-presented peptide epitope of the Wilms tumor antigen-1 (WT1) can selectively kill immature human leukemia progenitor and stem cells in vitro. In this study we have used retroviral gene transfer to introduce a WT1-specific T-cell receptor (TCR) into T lymphocytes obtained from patients with leukemia and from healthy donors. TCR-transduced T cells kill leukemia cells in vitro and display WT1-specific cytokine production. Intravenous injection of TCR-transduced T cells into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice harboring human leukemia cells resulted in leukemia elimination, whereas transfer of control T cells transduced with an irrelevant TCR was ineffective. The data suggest that adoptive immunotherapy with WT1-TCR gene-modified patient T cells should be considered for the treatment of leukemia.

Animals↗
PubMed2005

CY15, a malignant histiocytic tumor that is phenotypically similar to immature dendritic cells.

The origin and pathogenesis of histiocytic malignancies and the biology of the tumor cells are poorly understood. We have isolated a murine histiocytic tumor cell line (CY15) from a BALB/c IFNgamma(-/-) mouse and characterized it in terms of phenotype and function. The morphology, as judged by electron microscopy, and the surface marker phenotype suggests that CY15 cells are similar to immature dendritic cells (CD11c (low), MHC II (low), CD11b(+), B7.1(+), B7.2(+), and CD40(+)). The cells form tumors in BALB/c mice and metastasize to spleen, liver, lung, kidney, and to a lesser extend to lymph nodes and bone marrow, as judged by the growth of green fluorescent protein transfected tumor cells in mice. CY15 cells are capable of actively taking up antigen (FITC-ovalbumin) and can stimulate T lymphocytes in an allogenic mixed lymphocyte reaction but less effectively than their normal counterparts (immature dendritic cells). They respond to interleukin 4 (IL-4) with up-regulation of CD11c. If stimulated with IFNgamma the cells up-regulate MHC II, CD40 B7.1, and B7.2. Lipopolysaccharide induces the cells to up-regulate B7.1 and B7.2 and to secrete tumor necrosis factor alpha and IL-12. Based on these data, CY15 is a dendritic cell-like tumor cell line and may serve as a transplantable tumor model for histiocytosis in humans.

Animals↗
PubMed2005

Redirecting human T lymphocytes toward renal cell carcinoma specificity by retroviral transfer of T cell receptor genes.

Adoptive T cell therapy of renal cell carcinoma (RCC) is limited by the difficulty in generating sufficient numbers of RCC-reactive T cells in vitro. To circumvent this problem, we cloned T cell receptor (TCR) alpha and beta chains from a tumor-infiltrating lymphocyte clone specific for an RCC tumor antigen and transferred the TCR into human T cell lines and primary T lymphocytes. Efficient TCR expression in primary T lymphocytes was obtained only with a mouse myeloproliferative sarcoma virus (MPSV)-based retroviral vector, not with a Moloney murine leukemia virus (MLV)-based vector, although both viral supernatants were similar in titer, as shown by analysis of copy number integration in transduced T cells. Reverse transcription-polymerase chain reaction analysis revealed a higher amount of TCR-encoding transcripts when T cells were transduced with the MPSV vector in comparison with the MLV vector, indicating that high TCR expression levels can be achieved by appropriate cis-regulatory vector elements. The biological activity of the transferred TCR was shown by specific lysis of RCC cells ((51)Cr release assay) and by interferon gamma and tumor necrosis factor alpha release (enzyme-linked immunosorbent assay) in an antigen-specific and HLA-A*0201-restricted fashion. Comparison of the redirected T lymphocytes with the original tumor-infiltrating lymphocyte clone revealed similar killing and cytokine secretion capabilities. The functional activity of TCR-redirected T lymphocytes was stable over time. The results demonstrate that use of an optimized retroviral vector yielded a high TCR transduction efficiency and stable and high TCR expression in primary human T lymphocytes and redirected their specificity toward RCC cells.

Carcinoma, Renal Cell↗
PubMed2004

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1.

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth.

Adenoviridae↗
PubMed2004

Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors.

Grafting T cells with new antigen specificity by T cell receptor (TCR) gene transfer could greatly facilitate adoptive T cell immunotherapy. Little is known about how two TCR on one T cell influence each other. Among other reasons, this is often due to the fact that only one TCR specificity is known. We have genetically generated murine dual TCR T cells (OT-I/P14), specific for ovalbumin(ova257) and lymphocyte choriomeningitis virus glycoprotein (gp33). These cells retain both specificities and can be stimulated by either antigenic peptide to proliferate and produce IFN-gamma. Even though one TCR (P14) is expressed at reduced levels on dual TCR T cells, the peptide sensitivity of these cells is similar to that of single TCR T cells of the same specificity. TCR down-modulation on dual TCR T cells depends primarily on binding of the specific ligand. Adoptively transferred dual TCR T cells suppress the growth of both B16-ova and B16-gp33 melanoma cells, regardless of the peptide used for in vitro activation. Taken together, despite a certain dominance of expression between two TCR on the same T cell, this need not necessarily have functional consequences.

Adoptive Transfer↗
PubMed2004

Non-physiological overexpression of the low density lipoprotein receptor (LDLr) gene in the liver induces pathological intracellular lipid and cholesterol storage.

BACKGROUND: Gene therapy of familial hypercholesterolemia (FH) requires successful transfer and lifelong expression of a functional low density lipoprotein receptor (LDLr) gene in the liver. Most of the vector systems currently employed for gene therapy use promoter elements which do not modulate transgene expression in a physiological manner. METHODS: To study the in vivo effects of constitutive LDLr gene expression in the absence of interfering immunological reactions we established a new mouse model which combines homozygous LDLr deficiency and severe combined immune deficiency (SCID). RESULTS: Adenovirus-mediated transfer and expression of the LDLr gene under the control of a commonly used virus-derived promoter (minimal CMV promoter) leads to prolonged reduction of serum cholesterol levels in LDLr-deficient SCID mice. During the first 10 days after gene therapy serum cholesterol drops to about 10% of pretherapeutic values. Serum cholesterol persists on this level for 2 weeks and then slowly starts to rise again. Four months after vector application serum levels have reached about 40% of pretherapeutic values. However, as early as 5 days after gene transfer, the histological analysis of liver sections revealed the formation of crystalline lipid/cholesterol deposits in the cytosol of hepatocytes. During the following 8 weeks the amount of crystals increased in size and density. The intracellular storage of lipid and cholesterol reduced cell viability and induced an accelerated loss of therapeutic DNA from mice livers as was shown in a comparative expression study employing a transgene with a different metabolic function (human alpha 1-antitrypsin). CONCLUSIONS: The non-physiological constitutive overexpression of an LDL receptor gene induces an imbalance between the speed of LDL uptake and metabolism which leads to pathological accumulation of lipids and cholesterol in hepatocytes. To protect cells from negative effects of LDLr overexpression, future vector design should consider the use of physiologically controlled expression elements.

Animals↗
PubMed2003

Transduction efficiency of MLV but not of HIV-1 vectors is pseudotype dependent on human primary T lymphocytes.

The success of several gene therapeutic approaches requires efficient transduction of human primary T lymphocytes. For this it is important to enhance the transduction efficiency, and this can be achieved by various means, mainly technical development of transduction procedures and use of different vectors and vector pseudotypes. We analyzed the transduction efficiency of an HIV-1 vector encoding enhanced green fluorescent protein (GFP) as a marker gene and pseudotyped with the envelopes of MLV-A, MLV-10A1, GaLV, RD114, and VSV for human primary T lymphocytes in comparison to an MLV vector pseudotyped with the same envelopes. Pseudotyping of the MLV vector with the envelopes of 10A1 and GaLV resulted in efficient transduction of preactivated human primary T lymphocytes (32.4% and 32.7% CD3+/GFP+ cells, respectively) while MLV-A (14.0%), RD114 (8.8%), and VSV (1.5%) envelopes were less efficient when using titrated vector stocks equilibrated to a multiplicity of infection of 1. In contrast, the HIV-1 vectors pseudotyped with these envelope proteins transduced preactivated T lymphocytes with similar efficiency (approx. 20% CD3+/GFP+ cells). Thereby, CD4+ and CD8+ T lymphocyte subpopulations were transduced at equivalent levels. The similar performance of the different HIV-1 vector pseudotypes may be due in part to the similar half-lives of the vector particles. Independently of the envelope used for pseudotyping neither the MLV nor the HIV-1 vectors yielded any significant transduction in nonactivated T lymphocytes (below 0.55% of GFP+ cells)

CD4-CD8 Ratio↗
PubMed2003

Retroviral vectors for high-level transgene expression in T lymphocytes.

Efficient expression of genes transferred by retroviral vectors is a prerequisite for gene therapy, especially when the biological effect depends on the amount of transgene product. High-level gene expression is desirable for several gene therapy approaches involving T lymphocytes. We evaluated standard retroviral vectors with cis-regulatory control elements of the Moloney murine leukemia virus (Mo-MLV) with or without the human T cell-specific CD2 enhancer. For comparison, vectors containing the long terminal repeat (LTR) of myeloproliferative sarcoma virus (MPSV) and an improved 5' untranslated region were used (MP71 vectors), with or without the woodchuck hepatitis virus posttranscriptional regulatory element (PRE). All vectors expressed the enhanced green fluorescent protein (GFP) to measure transgene expression. In mouse T cells MP71 vectors with and without the PRE yielded an up to 10-fold higher expression level compared with the Mo-MLV-based vectors currently used for gene transfer into T lymphocytes. A high multiplicity of infection (MOI) of standard Mo-MLV vectors could not reach expression levels obtained with a low MOI of MP71 vector. Ex vivo-transduced mouse T lymphocytes maintained the vector-dependent differences in level of transgene expression in Rag-1-deficient mice when adoptively transferred. In four human T cell lines and human primary T lymphocytes MP71 vectors yielded an up to 75-fold higher GFP expression level in comparison with the standard Mo-MLV vector. In contrast to mouse T cells, the integration of the PRE into MP71 vectors induced in human T cells a further significant increase in transgene expression level. Southern blot analysis of CEM T cells revealed that the superior performance of MP71 vectors was not due to a higher rate of viral integration. In summary, MP71 vectors are useful tools for stable, high-level gene expression in T lymphocytes, for example, in the expression of T cell receptor genes.

3T3 Cells↗
PubMed2003

Overexpression of NPM-ALK induces different types of malignant lymphomas in IL-9 transgenic mice.

Anaplastic large-cell lymphoma (ALCL) comprises approximately 25% of all non-Hodgkin lymphomas (NHL) in children and young adults, and up to 15% of high-grade NHL in older patients. Over 50% of these tumours carry the translocation t(2;5)(p23;q35). The result of this translocation is the fusion of the nucleophosmin (NPM) gene to the anaplastic lymphoma kinase (ALK) gene. The resulting hybrid protein contains the ALK catalytic domain that consequently confers transforming potential, which contributes to the pathogenesis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM-ALK, was inserted into the retrovirus vector pLXSN and transduced into mouse bone marrow progenitors. These cells were subsequently used in a bone marrow transplant with the aim of reconstituting the haematopoietic compartments of lethally irradiated recipients. IL-9 transgenic mice were chosen as the animal model system, because dysregulated expression of the IL-9 gene in transgenic mice results in the sporadic development of spontaneous thymic lymphomas. Moreover, IL-9 is known to be expressed in cases of human ALCL. We used 15 IL-9 transgenic mice and eight corresponding wild-type mice (FVB/N) and transplanted them with NPM/ALK infected bone marrow cells. Eight IL-9 transgenic mice, serving as a control group, received pLXSN (vector only)-infected marrow. Reconstituted mice developed NPM-ALK-positive lymphomas, including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PC), and plasmoblastic/anaplastic diffuse large-B-cell lymphoma after about 19-20 weeks. The combined overexpression of NPM-ALK and IL-9 led to the transformation of murine lymphoid cells with accelerated and enhanced development of T-LB in 46% of the mice, which only very rarely occurs in IL-9 transgenic mice only. Of the 15 animals, five (33%) developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with anaplastic/plasmoblastic diffuse large-B-cell lymphoma on the basis of morphology, a characteristic growth pattern and ALK expression.

Animals↗
PubMed2003

Construction, rescue, and characterization of vectors derived from ovine atadenovirus.

Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

Adenoviridae↗