Lentiviruses infect both dividing and nondividing cells. In in pHP. The ability of this vector system to transduce dividthis study we characterized a lentiviral vector system coning and nondividing cell in vitro and in vivo was also demsisting of a packaging vector (pHP) and a transducing veconstrated. Compared with a Moloney murine leukemia tor (pTV) derived from a recombinant human immunodefivirus (MLV) vector, the HP/TV vectors transduced human ciency virus type 1 (HIV-1). In pHP, the long terminal muscle-, kidney-, liver-derived cell lines and CD34 + primary repeats (LTRs), the 5Ј untranslated leader and portions of hematopoietic progenitor cells more efficiently. Although the env and nef genes were deleted. The leader sequence the levels of the pTV transgene expression were high soon of pHP was substituted with a modified Rous sarcoma virus after transduction, the expression tended to decrease with (RSV) 59 bp leader containing a mutated RSV gag AUG time due either to the loss of proviral DNA or to the inactiand a functional 5Ј splice site. The pHP construct was vation of promoter activity, which was found to be cell typefound to direct Gag-Pol synthesis as efficiently as wild-type dependent. Analyses of extrachromosomal DNA showed HIV-1. The pTV construct contains sequences required for that the unintegrated proviral DNA of lentiviral vectors sur-RNA packaging, reverse transcription and integration, but vived much longer than that of the retroviral vectors. We lacks viral genes. Co-transfection of pHP, pTV and a vesdemonstrate that the HP/TV vector is capable of high icular stomatitis virus G (VSV-G) envelope plasmid proefficiency transduction and that long-term expression of duced vectors at titers of 10 5 -10 6 transducing units per lentiviral vectors is dependent on target cell type, the milliliter in 48 h. Replication-competent virus (RCV) was internal promoter and the transgene itself in the transducnot detected when deletions were made in the env gene ing vector.
There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65–90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.
One important issue using cells as therapeutics is targeted delivery. Engineering cell surfaces to improve delivery efficiency is thus of great interest. Here we report a simple, efficient and effective way to modify the cell surface with target-specific ligands, i.e., DNA aptamers, while minimizing the effects on the modified cells. We demonstrated that after incubating with lipo-aptamer probes (shown in expansion), immune cells (red) recognize cancer cells (blue) in the cell mixture, and kill cancer cells.
Lentiviral vectors have gained much attention in recent years mainly because they integrate into nondividing host-cell genomes. For clinical applications, a safe and efficient lentiviral vector system is required. Previously, we have established a human immunodeficiency virus type 1 (HIV-1)-derived three-plasmid lentiviral vector system for viral vector production which includes a packaging vector pHP, a transducing vector pTV, and an envelope-encoding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 10(5)-10(6) infectious units per milliliter in 24 h. Here we have extensively modified long terminal repeats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) enhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% wild-type vector efficiency. Further deletion of 3' U5 impaired vector function; however, this problem was solved by replacing the 3' U5 with bovine growth hormone polyadenylation (bGHpA) sequence. The pTV vector containing all these modifications including the 5' promoter substitution, the 3' U3 deletion, and the substitution of 3' U5 with bGHpA exhibited a self-inactivating (SIN) phenotype after transduction, transduced both dividing and nondividing cells at similar efficiencies, and produced vector titers twice as high as that of the wild-type construct. Thus, both safety and efficacy of the HP/TV vector have been improved by these LTR modifications. Further deletion of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has critical roles in vector function.
BackgroundCTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma.MethodsCTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student’s t-test.ResultsAll melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts.ConclusionsOur studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.
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