Peng H. Tan

Inhibition of NF-κB and Oxidative Pathways in Human Dendritic Cells by Antioxidative Vitamins Generates Regulatory T Cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and α-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2α, NF-κB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4+CD45RO, CD4+CD45RA, and CD4+CD25− subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-β. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.

Function of indoleamine 2,3‐dioxygenase in corneal allograft rejection and prolongation of allograft survival by over‐expression

Indoleamine 2,3‐dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over‐expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up‐regulated by IFN‐γ and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over‐expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up‐regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over‐expression in donor cornea was found to significantly extend survival of allografts.

Antibody targeted gene transfer to endothelium

One of the drawbacks of the currently available vectors for gene therapy is the lack of selectivity in gene delivery. We have therefore investigated a strategy to generate immunoliposomes to target non‐viral vectors to cell surface receptors on endothelium.

Aspirin-Treated Human DCs Up-Regulate ILT-3 and Induce Hyporesponsiveness and Regulatory Activity in Responder T Cells

Mature dendritic cells (mDCs) are potent antigen presenting cells, but immature DCs (iDCs) have been shown to have reduced antigen stimulatory capacity. Different strategies have been investigated to augment the tolerogenic capacity of dendritic cells (DCs). We demonstrate that in aspirin‐treated human DCs, there is reduced expression of CD1a, HLA‐DR and CD86, up‐regulation of ILT‐3 expression and marginal increases in PDL‐1. Aspirin‐treated DCs are partially resistant to phenotypic changes following maturational stimuli, such as lipopolysaccharide (LPS) or TNFα, IL‐1α and PGE2. Aspirin‐treated DCs demonstrate normal endocytic function, but have a reduced ability to stimulate allogeneic T cells, which is comparable to iDCs. Furthermore, they induce hyporesponsiveness and regulatory activity in responder naïve and memory T cells; for naïve T cells this is achieved more quickly and efficiently than with iDCs. We investigated the mechanism of this regulatory activity and found that both cell‐cell contact and inhibitory cytokine activity are involved, although no one cytokine predominates in importance. Blocking ILT‐3 or IL‐12 does not diminish the capacity of these DCs to induce regulation or Foxp3 expression on the regulatory T cells. Results demonstrate that aspirin‐treated DCs display tolerogenic potential, which is of interest in their therapeutic potential in reducing chronic allograft rejection.

Transferrin receptor-mediated gene transfer to the corneal endothelium.

Background. The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. Methods. Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. Results. Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eightfold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. Conclusions. Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.

Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers

INTRODUCTION The adhesion molecules P- and E-selectin are expressed on activated endothelial cells, and are good targets for gene therapy aimed at inflammatory disease. We have therefore investigated the potential of targeting PAMAM dendrimers, a non viral vector system, to cells expressing P/E-selectin using a monoclonal antibody that recognises these molecules. MATERIALS AND METHODS We used biotin and avidin to cross-link anti E/P-Selectin monoclonal antibody to pre-formed Superfect-DNA complexes that were then used to transfect reporter genes to CHO cells expressing E-Selectin, cytokine-activated primary Human Saphenous Vein Endothelial Cells (HSVEC) and whole vein segments. RESULTS The use of the anti E/P-Selectin antibody increased the transfection efficiency in CHO-E cells, activated HSVEC and saphenous vein segments ex vivo. We also showed that the antibody improved the binding of the complexes onto cells as well as the internalisation kinetics. DISCUSSION We demonstrate here that by attaching antibodies onto PAMAM dendrimers the efficiency of transfection can be significantly improved in cells or tissues expressing the receptor. This technology has potential in the treatment of cardiovascular disease by gene therapy but can also be used with different antibodies to target other diseased cells or tissues.

Manipulation of indoleamine 2,3 dioxygenase; a novel therapeutic target for treatment of diseases

Background: The discovery of indoleamine 2,3-dioxygenase (IDO) as a modulator for the maintenance of fetomaternal immuno-privileged state has been heralded as a significant step in further defining the role of IDO in immunobiology. IDO is an IFN-inducible, intracellular enzyme that catalyzes the initial and rate-limiting step in the degradation of the essential amino acid, tryptophan. It has been suggested that IDO has the capacity to regulate the immune system via two discrete mechanisms; firstly the deprivation of tryptophan, which is essential for T cell proliferation and via the cytotoxic effects of tryptophan metabolites on TH1 cell survival. Methods: The sources of information used to prepare the paper are published work on Pubmed/Medline. In this review, we examine the therapeutic role of modulating IDO activity a variety of disease states including tumour tolerance, chronic infection, transplant rejection, autoimmunity and asthma. We propose that IDO represents a novel therapeutic target for the treatment of these diseases. We also explore the diverse strategies which are being employed, either to augment or to inhibit IDO activity in order to modify various disease processes. The limitations associated with these strategies are also scrutinized.

Effect of vectors on human endothelial cell signal transduction: implications for cardiovascular gene therapy.

Objective—Endothelium is an important target for gene therapy. We have investigated the effect of viral and nonviral vectors on the phenotype and function of endothelial cells (ECs) and developed methods to block any activation caused by these vectors. Methods and Results—Transduction of ECs with viral vectors, including adenovirus, lentiviruses, and Moloney murine leukemia virus, can induce a pro-inflammatory phenotype. This activation was reduced when nonviral vectors were used. We demonstrate that after transduction there is upregulation of dsRNA-triggered antiviral and PI3K/Akt signaling pathway. Blockade of the NF&kgr;B, PI3-K, or PKR signaling pathways all operated to inhibit partially virally induced activation, and inhibition of both PKR and PI3-K pathways totally blocked EC activation. Furthermore, inhibition of IFN-α/β in addition to PI3-K was effective at preventing EC activation. Conclusions—Viral vectors, although efficient at transducing ECs, result in their activation. Blockade of the signaling pathways involved in viral activation may be used to prevent such activation.

Tolerant t cells display impaired trafficking ability

Based on our previous observation that anergic T lymphocytes lose their migratory ability in vitro, we have proposed that anergic T cells are retained in the site where they have been generated to exert their regulatory function. In this study we have analyzed T lymphocyte trafficking and motility following the induction of tolerance in vivo. In a model of non‐deletional negative vaccination to xenoantigens in which dendritic cells (DC) localize to specific lymphoid sites depending on the route of administration, tolerant T cells remained localized in the lymph nodes colonized by tolerogenic DC, while primed T cells could traffic efficiently. Using an oral tolerance model that enables the ‘tracking’ of ovalbumin‐specific TCR‐transgenic T cells, we confirmed that T cells lose the ability to migrate through syngeneic endothelial cell monolayers following tolerance induction in vivo. Finally, we show that tolerant T cells (both in vitro and ex vivo) can inhibit migration of responsive T cells in an antigen‐independent manner. Thus, hyporesponsive T cells localize at the site of tolerance induction in vivo, where they exert their anti‐inflammatory properties. In physiological terms, this effect is likely to render immunoregulation a more efficient and controllable event.

Mesenchymal stem cells differentially mediate regulatory T cells and conventional effector T cells to protect fully allogeneic islet grafts in mice

Aims/hypothesisLimited information is available on the cellular interactions between regulatory T (Treg) cells and mesenchymal stem cells (MSCs). In particular, a direct effect of MSCs on the survival and proliferation of Treg cells has not been demonstrated.MethodsWe investigated the effects of MSCs on effector T (Teff) cells and Treg cells, and the molecular mechanisms involved in the distinct regulation of these two cell populations by MSCs in vivo and in vitro.ResultsWe show that MSCs are capable of selectively suppressing Teff cells and fostering the generation of Treg cells. Teff cells, but not Treg cells, fail to respond to IL-2 and undergo profound apoptosis in the presence of MSCs. The differential regulations of these two T cell subsets by MSCs are associated with their distinct expressions of CD25, with MSCs specifically reducing the expression of CD25 on Teff and sparing Treg cells intact. In vivo, the administration of MSCs significantly delays the rejection of allogeneic islet grafts in adaptive transferred recipients by favouring the induction of Treg cells. In this model, MSCs inhibit the proliferation and development of alloreactive Teff but potently enhance the induction of Treg cells.Conclusions/interpretationWe demonstrate that MSCs are capable of regulating Teff and Treg cells differentially in vitro. MSCs inhibit Teff cells by inducing apoptosis and impairing the proliferative response to IL-2 in Teff cells, but favour the survival and expansion of Treg cells. This result is further demonstrated in mice that have undergone allogeneic islet transplantation, in which MSCs suppress alloreactive Teff cells while favouring the induction of Treg cells, thus protecting the islet allografts from rejection.