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Distinct strategies are required to suppress antigen-specific responses to genetically modified keratinocytes and fibroblasts.

ABSTRACT: Keratinocytes and fibroblasts are potential targets of gene/cell therapy for genodermatoses. Immune elimination of genetically modified cells, however, presents a major impediment to effective therapy. Using ex vivo approaches to gene transfer, we have previously shown that expression of an antigen by either cell type in skin induces immune rejection of transplanted cells, although the nature of immune responses induced by these two cell types are distinct. In this study, we explore the efficacy of local immunosuppressive strategies to divert destructive immune responses from genetically modified fibroblast and keratinocytes. Expression of CTLA4Ig and, to a lesser extent, PDL1, by antigenic fibroblasts protected them from immune rejection resulting in long-term graft survival (>18 weeks). Similar treatment was not effective for antigenic keratinocytes. Long-term protection of transgenic keratinocytes was achieved through transient blockade of CD40/CD154 interactions during the first 2 weeks of cell transplantation. Although neither of these strategies induced antigen-specific tolerance, they were sufficient to prevent rejection of genetically modified cells. These results indicate that different strategies are required to protect antigenic cell types even within the same tissue. Moreover, induction of antigen-specific tolerance is not a necessary requirement for long-term survival of genetically modified skin cells.

SUBMITTER: Ghazizadeh S

PROVIDER: S-EPMC3255584 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Distinct strategies are required to suppress antigen-specific responses to genetically modified keratinocytes and fibroblasts.

Ghazizadeh Soosan S Huang Li T LT Zhang Weibing W

Molecular therapy : the journal of the American Society of Gene Therapy 20111011 1

Keratinocytes and fibroblasts are potential targets of gene/cell therapy for genodermatoses. Immune elimination of genetically modified cells, however, presents a major impediment to effective therapy. Using ex vivo approaches to gene transfer, we have previously shown that expression of an antigen by either cell type in skin induces immune rejection of transplanted cells, although the nature of immune responses induced by these two cell types are distinct. In this study, we explore the efficacy ...[more]

PMID: 21988876

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Project description:BackgroundThe Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing).ResultsIn this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts.ConclusionsThe methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.

Project description:Background & aimsHBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs.MethodsThe replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs.ResultsPatient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C.ConclusionsThe infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening.Lay summaryThe hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

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Distinct strategies are required to suppress antigen-specific responses to genetically modified keratinocytes and fibroblasts.

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Distinct strategies are required to suppress antigen-specific responses to genetically modified keratinocytes and fibroblasts.

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