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  • Gene delivery to the corneal endothelium.

    12 December 2017

    Gene transfer to the corneal endothelium has potential for modulating rejection of corneal grafts. It can also serve as a convenient and useful model for gene therapy of other organs. In this article we review the work carried out in our laboratory using both viral and nonviral vectors to obtain gene expression in the cornea.

  • Auto-antibody production and glomerulonephritis in congenic Slamf1-/- and Slamf2-/- [B6.129] but not in Slamf1-/- and Slamf2-/- [BALB/c.129] mice.

    12 December 2017

    Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.

  • A CIITA-independent pathway that promotes expression of endogenous rather than exogenous peptides in immune-privileged sites.

    8 December 2017

    A CIITA-independent pathway of MHC class II expression has been found in the eye and the brain, both immune-privileged sites. Although corneal endothelial cells were unable to express MHC class II in response to IFN-gamma alone, these cells readily expressed MHC class II molecules via a CIITA-independent pathway when triggered by simultaneous exposure to IFN-gamma and TNF-alpha. CIITA-independent expression of MHCclass II molecules enabled corneal endothelial cells to present cytosolic, but not endosomal, ovalbumin (OVA) to OVA-primed T cells. To determine whether CIITA-independent expression of MHC class II is relevant in vivo, minor H-only-incompatible corneal allografts prepared from CIITA knockout (KO) mice, MHC class II KO mice or wild-type donors were placed in eyes of normal mice. Cornea allografts from wild-type and CIITA KO mice suffered similar rejection fates, whereas far fewer class II-deficient corneas were rejected. In addition, MHC class II-bearing macrophages were observed in cuprizone-induced inflammatory and demyelinating brain lesions of CIITA KO mice. We conclude that class II expression via the CIITA-independent pathway enhances the vulnerability to rejection of corneal grafts expressing minor antigens. The potential relevance of CIITA-independent MHC class II expression at immune-privileged sites is discussed in relation to tolerance to strong autoantigens.

  • Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity.

    12 December 2017

    Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

  • Lipoadenofection-mediated gene delivery to the corneal endothelium: prospects for modulating graft rejection.

    12 December 2017

    BACKGROUND: Gene transfer to the corneal endothelium has potential for the prevention or reversal of corneal allograft rejection. Previous work has examined adenoviral vectors for gene transfer to endothelium. These have a number of theoretical and practical disadvantages, both for experimental and clinical applications. We have therefore used lipoadenofection, in which plasmid DNA is delivered using a combination of liposomes and adenovirus, to transfer marker genes to the cornea. METHODS: Corneas were obtained from New Zealand White rabbits and cultured ex vivo using standard conditions. The corneas were transfected using either lipofection or lipoadenofection with plasmids encoding marker genes. The efficiency of gene transfer and the location and kinetics of gene expression were determined. We also investigated the delivery of a gene construct containing an inducible promoter that is activated by tumor necrosis factor (TNF), to determine whether expression of the relevant genes could be controlled by exogenous factors such as cytokines. RESULTS: This study shows that gene expression is limited to the endothelium and that expression is transient. Furthermore, we have shown that expression of a gene controlled by an inducible promoter only occurs when TNF is present. CONCLUSIONS: These data indicate that lipofection is an efficient method to transfer therapeutic genes to the corneal epithelium, and that it can be used to transfer constructs that utilize an inducible promoter controlled by TNF. As TNF is present in the aqueous humor during allograft rejection, and this is in contact with the corneal endothelium, this has the potential to restrict expression of a therapeutic gene to rejection episodes in the cornea.

  • Co‐Administration of Co‐Stimulatory Moieties

    17 November 2017

    Covering all aspects of vaccine research and development in one volume, this authoritative resource takes a comprehensive and systematic approach to the science of vaccinology focusing not only on basic science, but also on the many stages ...

  • Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.

    12 December 2017

    Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

  • A method for determining the cytoprotective effect of catalase in transiently transfected cell lines and in corneal tissue.

    12 December 2017

    Both when developing gene constructs for therapeutic purposes and when testing the biological function of proteins, it would be convenient to use cells or tissues that have been transiently transfected with the gene of interest. However, determining the protective effects of transient gene expression is complicated by a low transfection efficiency, resulting in only a minority of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this study we have developed a convenient technique for determining cell death in transiently transfected vascular endothelial cell monolayers and in corneal tissue. Vascular endothelial cells were cotransfected with human catalase cDNA and the lacZ gene encoding beta-galactosidase, under conditions in which cells expressing beta-galactosidase also expressed catalase. By assaying release of beta-galactosidase upon cell death, it was possible to show that catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was adapted to demonstrate the protective effects of catalase transfection on hydrogen peroxide-mediated injury of intact corneal endothelium under ex vivo culture conditions. This assay should also be useful for characterizing the cytoprotective effects of other genes in transient transfection systems.

  • Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells.

    6 December 2017

    Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein-protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.

  • Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    12 December 2017

    Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.