Transgene-specific Immune Responses Research Articles

412. Cyclosporine Inhibits the Induction of GFP Specific Immune Responses after Transplantation of Lentivirally Transduced GFP Expressing Hematopoietic Stem Cells in Dogs

Green fluorescent proteins have been widely used to monitor gene transfer and expression following lentiviral and oncoretroviral transduction of baboon and canine repopulating hematopoietic cells. Recent studies in our laboratory have shown a complete disappearance of GFP-containing cells following transplantation of lentivirally transduced repopulating cells in several baboons. The complete disappearance of genetically modified cells was shown to be mediated by a transgene specific immune response. We have also observed a complete disappearance of GFP marked cells following transplantation of lentivirally transduced hematopoietic cells in dogs. To identify a possible role the immune system may have in the disappearance of these cells, eight dogs were preconditioned with ablative irradiation and were transplanted with a lentiviral vector containing the GFP transgene. Blood samples were obtained twice a week for up to 800 days and the GFP transgene product was measured by flow cytometry in blood leukocytes. Six dogs showed stable GFP marking in blood leukocytes over 800 days demonstrating engraftment of transgene expressing cells. However, two dogs began to lose marking between 30 and 50 days post-transplant. To determine whether an immune response to GFP was responsible for the disappearance of gene modified cells in dogs that exhibited a decrease in GFP marking, peripheral blood leukocytes were isolated by a Ficoll gradient and stimulated in vitro for 5 days using GFP peptides. Immune responses were detected by measuring intracellular levels of TNFa using flow cytometry. Dogs that exhibited a decrease in GFP marking developed potent immune responses in vitro to the transgene product GFP as shown by an increase in GFP specific TNFa production (p<0.05) when compared to non-transplanted controls. To determine whether cyclosporine could prevent disappearance of GFP marked cells, six dogs were treated with cyclosporine following transplantation of lentivirally transduced hematopoietic cells. Dogs treated with cyclosporine post- transplant showed stable GFP marking in blood leukocytes over 800 days. However, two dogs that were not treated with cyclosporine exhibited a complete loss of GFP marking in blood leukocytes. Furthermore, potent immune responses were detected in dogs that had not received post-transplant cyclosporine treatment (n = 2; p<0.05). Our data suggest that cyclosporine prevents immunoactivation against transgene products following bone marrow transplantation using lentivirally modified cells as indicated by stable GFP marking. These findings should have important implications for the development of stem cell gene therapy protocols for patients with generic diseases.

657. Immune Responses to Transgene Expression Targeted to Keratinocytes

Transgene-specific immune responses impose a major hurdle to effective implementation of clinical gene therapy, especially in situations where neoantigen is expressed. Previously, in our studies of cutaneous gene therapy, we showed that neoantigen expression leads to a potent immune response and rejection of the retroviral-transduced epidermal cells in normal mice. As cutaneous gene therapy will often require prolonged transgene expression, we seek a strategy to avoid this host response. One such strategy utilizes the fact that antigen presentation by non-professional antigen presenting cells (APCs), including keratinocytes, may result in T cell unresponsiveness. To explore this concept, the dorsal skin of immunocompetent mice was transduced in vivo with 2 × 107 transducing units of either standard retroviral vector encoding GFP or retroviral vectors in which GFP expression is targeted to the suprabasal layers of epidermis. Initial GFP expression in both groups of animals was confirmed at 1-week post transduction by fluorescent stereo microscopy. Examination at 3 weeks however, indicated loss of GFP expression in both groups of transduced mice coinciding with the generation of significant levels of anti-GFP antibody. Although expression was directed to epidermal keratinocytes, the acute loss of transduced cells may have resulted from uptake of GFP entrapped in the viral particle by professional APCs, which are abundant in skin. This possibility is currently being examined with an ex vivo model of cutaneous gene transfer in which transgene expression will be confined solely to keratinocytes and transplanted to immunocompetent mice.

Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines.

Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning

AbstractLentiviral vectors are increasingly being used for transferring genes into hematopoietic stem cells (HSCs) due to their ability to transduce nondividing cells. Whereas results in in vitro studies and the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) model have been highly encourgaging, studies in large animals have not confirmed the superior transduction of HSCs using lentiviral vectors versus oncoretroviral vectors. In contrast to the stable gene marking we have consistently achieved with oncoretroviral vectors in animals that received myeloablative conditioning, we observed the complete disappearance of genetically modified enhanced green or yellow fluorescent protein–expressing cells in 5 baboons that received transplants of HSCs transduced with lentiviral vectors alone or in combination with oncoretroviral vectors. Immune responses to transgene products have been found to be involved in the disappearance of gene-modified cells after nonmyeloablative conditioning. Thus, we examined whether the disappearance of genemodified cells after ablative conditioning may be due to an immune response. In 4 of 5 animals, cytotoxic T lymphocytes specific for the transgene protein were readily detected, demonstrating that immune reactions were responsible for the disappearance of the gene-marked cells in the animals. In summary, we report the induction of transgene-specific immune responses after transplantation of lentivirally transduced repopulating cells in a myeloablative setting.

Oral Plasmid DNA Delivery Systems for Genetic Immunisation

The use and optimisation of plasmid DNA delivery systems for the purposes of eliciting transgene specific immune responses to orally administered DNA encoded antigen represents a significant challenge. Here, we have outlined a multicomponent polymer modified liposomal delivery system that offers potential for oral administration of plasmid DNA. It is shown that the polymer/liposome formulated DNA is able to elicit markedly enhanced transgene specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen. This is discussed with reference to recent publications and the potential of plasmid DNA delivery systems for the purposes of genetic immunisation, as reported in selected literature, is assessed.

Liposome/DNA complexes coated with biodegradable PLA improve immune responses to plasmid encoding hepatitis B surface antigen.

We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen-presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 microg of the plasmid pRc/CMV-HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene-specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T-cell proliferation and cytokine production upon re-stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the subcutaneous route. In addition, the polymer-modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses (n=5, P<0.03). Our observations provide 'proof of principle' for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.

Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model.

The in vivo persistence of gene-modified cells can be limited by host immune responses to transgene-encoded proteins. In this study we evaluated in a nonhuman primate model whether the administration of a nonmyeloablative regimen consisting of low-dose total-body irradiation with 200 cGy followed by immunosuppression with mycophenolate mofetil and cyclosporin A for 28 and 35 days, respectively, could be used to facilitate persistence of autologous gene-modified T cells when a transgene-specific immune response had already been established or to induce long-lasting tolerance in unprimed recipients. Two macaques (Macaca nemestrina) received infusions of T cells transduced to express either the enhanced green fluorescent protein and neomycin phosphotransferase genes or the hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes. In the absence of immunosuppression, both macaques developed potent class I major histocompatibility complex-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified T cells and that persisted long term as memory CTL. Treatment with the nonmyeloablative regimen failed to abrogate preexisting memory CTL responses but interfered with the induction of transgene-specific CTL and facilitated in vivo persistence of gene-modified cells in an unprimed host. However, sustained tolerance to gene-modified T cells was not achieved with this regimen, indicating that further modifications will be required to permit sustained persistence of gene-modified T cells.