Patricia Yotnda

Efficient infection of primitive hematopoietic stem cells by modified adenovirus.

Almost all studies of adenoviral vector-mediated gene transfer have made use of the adenovirus type 5 (Ad5). Unfortunately, Ad5 has been ineffective at infecting hematopoietic progenitor cells (HPC). Chimeric Ad5/F35 vectors that have been engineered to substitute the shorter-shafted fiber protein from Ad35 can efficiently infect committed hematopoietic cells and we now show highly effective gene transfer to primitive progenitor subsets. An Ad5GFP and Ad5/F35GFP vector was added to CD34+ and CD34−lineage− (lin−) HPC. Only 5–20% of CD34+ and CD34−lin− cells expressed GFP after Ad5 exposure. In contrast, with the Ad5/F35 vector, 30–70% of the CD34+, 50–70% of the CD34−lin− and up to 60% of the CD38− HPC expressed GFP and there was little evident cellular toxicity. Because of these improved results, we also analyzed the ability of Ad5/F35 virus to infect the hoechst negative ‘side population’ (SP) of marrow cells, which appear to be among the very earliest multipotent HPC. Between 51% and 80% of marrow SP cells expressed GFP. The infected populations retained their ability to form colonies in two short-term culture systems, with no loss of viability. We also studied the transfer and expression of immunomodulatory genes, CD40L (cell surface expression) and interleukin-2 (secreted). Both were expressed at immunomodulatory levels for >5 days. The ability of Ad5/F35 to deliver transgenes to primitive HPC with high efficiency and low toxicity in the absence of growth factors provides an improved means of studying the consequences of transient gene expression in these cells.

Use of a Chimeric Adenovirus Vector Enhances BMP2 Production and Bone Formation

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Induction of Therapeutic T-Cell Responses to Subdominant Tumor-associated Viral Oncogene after Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine

The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication- incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/Kb mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/Kb mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.

Autologous Antileukemic Immune Response Induced by Chronic Lymphocytic Leukemia B Cells Expressing the CD40 Ligand and Interleukin 2 Transgenes

Although the B cells of chronic lymphocytic leukemia (B-CLL cells) express both tumor-specific peptides and major histocompatibility complex (MHC) class I antigens, they lack the capacity for costimulatory signaling, contributing to their protection against host antitumor immunity. To stimulate CLL-specific immune responses, we sought to transfer the human CD40 ligand (hCD40L) gene to B-CLL cells, using an adenoviral vector, in order to upregulate costimulating factors on these cells. Because efficient gene transduction with adenoviral vectors requires the expression of virus receptors on target cells, including the coxsackievirus B-adenovirus receptors (CAR) and alpha(v) integrins, we cocultured B-CLL cells with human embryonic lung fibroblasts (MRC-5 line). This exposure led to increased expression of integrin alpha(v)beta3 on B-CLL cells, which correlated with higher transduction rates. Using this novel prestimulation system, we transduced B-CLL cells with the hCD40L gene. The Ad-hCD40L-infected cells had higher expression of B7 molecules and induced activation of autologous T cells in vitro, but these T cells could not recognize parental leukemic cells. By contrast, an admixture of Ad-hCD40L-positive cells and leukemic cells transduced with the human interleukin 2 (IL-2) gene produced greater T cell activation than did either immunostimulator population alone. Importantly, this combination generated autologous T cells capable of specifically recognizing parental B-CLL cells. These findings suggest that the combined use of genetically modified CD40L-expressing B-CLL cells in combination with IL-2-expressing B-CLL cells may induce therapeutically significant leukemia-specific immune responses.

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells.

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non‐osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20‐17. However, human bone marrow stromal cells cultured with 1 μM dexamethasone for four days, or further stimulated to become osteoblast‐like cells with 50 μg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 μM dexamethasone was reduced if the cells were not given 50 μg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells. J. Cell. Biochem. 82: 11–21, 2001.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Crosstalk from Non-Cancerous Mitochondria Can Inhibit Tumor Properties of Metastatic Cells by Suppressing Oncogenic Pathways

Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK- cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with non-cancerous mitochondria. These results suggest the critical oncogenic regulation by mitochondrial-nuclear cross talk and highlights rectifying mitochondrial functional properties as a promising target in cancer therapy.

Transgenic expression of CD40L and interleukin-2 induces an autologous antitumor immune response in patients with non-Hodgkin's lymphoma

The malignant B cells of non-Hodgkins lymphoma (B-NHL cells) express peptides derived from tumor-specific antigens such as immunoglobulin idiotypes, and also express major histocompatibility complex antigens. However, they do not express co-stimulatory molecules, which likely contributes to their protection from host antitumor immunity. To stimulate NHL-specific immune responses, we attempted to transfer the human CD40 ligand (hCD40L) gene to B-NHL cells and enhance their co-stimulatory potential. We found that an adenoviral vector encoding human CD40L (AdhCD40L) was ineffective at transducing B-NHL cells because these cells lack the coxsackievirus B-adenovirus receptor and αv integrins. However, preculture of the B-NHL cells with the human embryonic lung fibroblast line, MRC-5, significantly up-regulated expression of integrin αvβ3 and markedly increased their susceptibility to adenoviral vector transduction. After prestimulation, transduction with AdhCD40L increased CD40L expression on B-NHL cells from 1.3±0.2% to 40.8±11.9%. Transduction of control adenoviral vector had no effect. Expression of transgenic human CD40L on these CD40-positive cells was in turn associated with up-regulation of other co-stimulatory molecules including B7-1/-2. Transduced B-NHL cells were now able to stimulate DNA synthesis of autologous T cells. However, the stimulated T cells were unable to recognize unmodified lymphoma cells, a requirement for an effective tumor vaccine. Based on previous results in an animal model, we determined the effects of combined use of B-NHL cells transduced with AdhCD40L and AdhIL2 vectors. The combination enhanced initial T-cell activation and generated autologous T cells capable of specifically recognizing and killing parental (unmodified) B-NHL cellsvia major histocompatibility complex–restricted cytotoxic T lymphocytes. These findings suggest that the combination of CD40L and IL2 gene-modified B-NHL cells will induce a cytotoxic immune responsein vivo directed against unmodified tumor cells. Cancer Gene Therapy (2001) 8, 378–387

Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment

Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.

KCa1.1 Potassium Channels Regulate Key Proinflammatory and Invasive Properties of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Background: Fibroblast-like synoviocytes participate in the pathogenesis of rheumatoid arthritis. Results: KCa1.1 is the major potassium channel on fibroblast-like synoviocytes from patients with rheumatoid arthritis, and blocking KCa1.1 channels perturbs the function of these cells. Conclusion: KCa1.1 channels play important regulatory roles in the function of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Significance: KCa1.1 channel are potential new therapeutic targets for rheumatoid arthritis. Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.