Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells

BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells that play important roles during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 derived lentiviral vectors (LVs) transduce DCs at high efficiency but their effects on DC functions have not been carefully studied. Modification of DCs using LVs may lead to important applications in transplantation, treatment of cancer, autoimmune and infectious diseases. RESULTS: Using DCs prepared from multiple blood donors, we report that LV transduction of DCs resulted in altered DC phenotypes and functions. Lentiviral transduction of DCs resulted in down-regulation of cell surface molecules including CD1a, co-stimulatory molecules CD80, CD86, ICAM-1, and DC-SIGN. DCs transduced with LVs displayed a diminished capacity to polarize naive T cells to differentiate into Th1 effectors. This impaired Th1 response could be fully corrected by co-transduction of DCs with LVs encoding interleukin-12 (IL-12), interferon-gamma (IFN-γ), or small interfering RNA (siRNA) targeting IL-10. CONCLUSIONS: DCs transduced with LVs in vitro displayed diminished Th1 functions due to altered DC phenotypes. Our study addresses an important issue concerning lentiviral infection and modification of DC functions, and provides a rational approach using LVs for immunotherapy

Overlapping enhancer/promoter and transcriptional termination signals in the lentiviral long terminal repeat

Oncoretrovirus, but not lentivirus, displays a high transcriptional readthrough activity in the 3' long terminal repeat (LTR) (Zaiss et al. J. Virol. 76, 7209–7219, 2002). However, the U3-deleted, self-inactivating (SIN) lentiviral LTR also exhibits high transcriptional readthrough activity. Since the canonical "core" polyadenylation signal (AAUAAA) of the lentivirus is located in the R-U5 region, the above finding suggests that additional RNA termination signals must be present in the U3 region. Insertion of alternative termination signals including panhuman T cell leukemia virus type I polyadenylation signal, a 3' end small intron, and a tertiary tRNA motif into the lentiviral SIN LTR did not restore the transcriptional termination function. Functional dissection of the U3 region revealed that 70–80% of the termination signals reside in the transcriptional control region within 124 nt overlapping NFκB, Sp1 and TATA binding sites. Serial deletion analysis of the transcriptional control region indicates that the lentiviral enhancer/promoter elements are essential to the RNA termination function. These results characterize the mechanism of lentiviral transcriptional readthrough, which addresses important fundamental and practical issue of RNA readthrough influencing lentiviral gene function and vector safety

Genetically Modified Dermal Keratinocytes Express High Levels of Transforming Growth Factor-β1

In an attempt to genetically modify cultured keratinocytes with transforming growth factor-β1 (TGF-β1), which has been proven to be one of the most important cytokines involved in wound healing, two constructs were made. One, designated pG3Z:K14-TGF-β1, is a plasmid in which the expression of TGF-β1 is driven by the keratin 14 promoter. The other, designated pLin-TGF-β1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expression. In both constructs, the deletion of a small fragment of the noncoding region of the TGF-β1 gene was made to differentiate the transcript from that for endogenously expressed TGF-β1. Different types of cells were transfected with the pG3Z:K14-TGF-β1 construct using the calcium phosphate method. The pLin-TGF-β1 construct was propagated in a retroviral packaging cell line and conditioned medium that contained high titers of the virus was used to transduce keratinocytes or other types of cells grown in standard culture. Northern analysis, used to evaluate the expression of TGF-β1 mRNA in the pG3Z:K14-TGF-β1 transfected keratinocyte Cl-177 cell line, showed a smaller TGF-β1 transcript compared with that endogenously expressed by dermal fibroblasts. The level of TGF-β1 protein evaluated by enzyme-linked immunosorbent assay was significantly higher in medium conditioned by either the K14-TGF-β1 transfected or the pLin-TGF-β1 transduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-β1 protein was unchanged in cultures of pG3Z:K14-TGF-β1 transfected nonkeratinocyte cells such as fetal and adult fibroblasts. Using the mink lung epithelial cell growth inhibition assay, we found an increase in TGF-β1 activity in conditioned medium from the pG3Z:K14-TGF-β1 transfected cells. To evaluate possible paracrine effects of the keratinocyte derived TGF-β1, a coculture system was established with pLin-TGF-β1 transduced keratinocytes grown in the upper chamber and dermal fibroblasts in the lower chamber. The results showed that TGF-β1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts. In summary, we demonstrate here that primary cultured keratinocytes can be genetically modified to express high levels of TGF-β1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds

Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications

Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-γ or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-γ-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-γ and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-γ-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-γ selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications

Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells

Dendritic cells (DCs) play a key role in innate and adaptive immunity but the access to sufficient amount of DCs for basic and translational research has been limited

Systemic Injection of RPE65-Programmed Bone Marrow-Derived Cells Prevents Progression of Chronic Retinal Degeneration

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases

Microarray meta-analysis database (M2DB): a uniformly pre-processed, quality controlled, and manually curated human clinical microarray database

<p>Abstract</p> <p>Background</p> <p>Over the past decade, gene expression microarray studies have greatly expanded our knowledge of genetic mechanisms of human diseases. Meta-analysis of substantial amounts of accumulated data, by integrating valuable information from multiple studies, is becoming more important in microarray research. However, collecting data of special interest from public microarray repositories often present major practical problems. Moreover, including low-quality data may significantly reduce meta-analysis efficiency.</p> <p>Results</p> <p>M²DB is a human curated microarray database designed for easy querying, based on clinical information and for interactive retrieval of either raw or uniformly pre-processed data, along with a set of quality-control metrics. The database contains more than 10,000 previously published Affymetrix GeneChip arrays, performed using human clinical specimens. M²DB allows online querying according to a flexible combination of five clinical annotations describing disease state and sampling location. These annotations were manually curated by controlled vocabularies, based on information obtained from GEO, ArrayExpress, and published papers. For array-based assessment control, the online query provides sets of QC metrics, generated using three available QC algorithms. Arrays with poor data quality can easily be excluded from the query interface. The query provides values from two algorithms for gene-based filtering, and raw data and three kinds of pre-processed data for downloading.</p> <p>Conclusion</p> <p>M²DB utilizes a user-friendly interface for QC parameters, sample clinical annotations, and data formats to help users obtain clinical metadata. This database provides a lower entry threshold and an integrated process of meta-analysis. We hope that this research will promote further evolution of microarray meta-analysis.</p

TNF-α Mediates Eosinophil Cationic Protein-induced Apoptosis in BEAS-2B Cells

<p>Abstract</p> <p>Background</p> <p>Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.</p> <p>Results</p> <p>To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-α). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-α antibody.</p> <p>Conclusion</p> <p>In conclusion, our results have demonstrated that ECP increased TNF-α production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.</p

Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells

Bacterial Delivery of Nuclear Proteins into Pluripotent and Differentiated Cells

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming