Endothelial Progenitor Cells (EPCs) as Gene Carrier System for Rat Model of Human Glioma

Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes

In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.</p> <p>Methods</p> <p>HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.</p> <p>Results</p> <p>FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4⁺and CD8⁺T cells able to produce IFN-γ and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated <it>Streptococcus pyogenes</it>. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4⁺CD25^highFoxp3⁺Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-γ.</p> <p>Conclusion</p> <p>The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.</p

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2neg B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8+ T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8+ T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8+ T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8+ T cells directly recognized HLA-A2+ pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8+ T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8+ T cells harvested from the blood of HLA-A2+ normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2+ cancer patients receiving therapeutic interventions, such as IL-12 gene therapy