[Show abstract][Hide abstract] ABSTRACT: In this study we evaluated stimulation of the angiotensin AT2-receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunised with myelin-oligodendrocyte-peptide (MOG) and treated for 4 weeks with C21 (0.3mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction of EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R-stimulation. C21 treated mice had a significantly better neurological score than vehicle treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R-stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and NO production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R-stimulation protects the myelin sheaths in autoimmune CNS inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.
[Show abstract][Hide abstract] ABSTRACT: The transfer of alloreactive Treg (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a non-depleting anti-CD4 antibody (aCD4). Here we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+) Foxp3(+) -expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+) Foxp3(+) cells from all culture conditions displayed complete demethylation of the TSDR region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon re-stimulation. Consequently, aCD4+TGF+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease (aGvHD) and especially skin transplant rejection. Thus addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells. This article is protected by copyright. All rights reserved.
European Journal of Immunology 12/2013; 43(12). DOI:10.1002/eji.201243292 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although mechanisms leading to brain-specific inflammation and T cell activation have been widely investigated, regulatory mechanisms of local innate immune cells in the brain are only poorly understood. In this study, to our knowledge we show for the first time that MHC class II(+)CD40(dim)CD86(dim)IL-10(+) microglia are potent inducers of Ag-specific CD4(+)Foxp3(+) regulatory T cells (Tregs) in vitro. Microglia differentially regulated MHC class II expression, costimulatory molecules, and IL-10 depending on the amount of IFN-γ challenge and Ag dose, promoting either effector T cell or Treg induction. Microglia-induced Tregs were functionally active in vitro by inhibiting Ag-specific proliferation of effector T cells, and in vivo by attenuating experimental autoimmune encephalomyelitis disease course after adoptive transfer. These results indicate that MHC class II(+)CD40(dim)CD86(dim)IL-10(+) microglia have regulatory properties potentially influencing local immune responses in the CNS.
The Journal of Immunology 10/2013; 191(11). DOI:10.4049/jimmunol.1203331 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Journal of Cerebral Blood Flow & Metabolism stands at the interface between basic and clinical neurovascular research, and features research on experimental, theoretical, and clinical aspects of brain circulation, metabolism and imaging. The journal is relevant to any physician or scientist with an interest in brain function, cerebrovascular disease, cerebral vascular regulation and brain metabolism, including neurologists, neurochemists, physiologists, pharmacologists, anesthesiologists, neuroradiologists, neurosurgeons, neuropathologists and neuroscientists.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 01/2013; 33(1):159. DOI:10.1038/jcbfm.2012.149 · 5.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3(EGFP) transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3(+) Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4(+) cells depleted of Foxp3(+) Tregs into RAG1(-/-) mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25(+) Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.Journal of Cerebral Blood Flow & Metabolism advance online publication, 12 September 2012; doi:10.1038/jcbfm.2012.128.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 09/2012; 33(1). DOI:10.1038/jcbfm.2012.128 · 5.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Innate immune receptors represent an evolutionarily ancient system that allows organisms to detect and rapidly respond to pathogen- and host-derived factors. TLRs are predominantly expressed in immune cells and mediate such a response. Although this class of pattern recognition receptors is involved in CNS disorders, the knowledge of ligands leading to activation of TLRs and to subsequent CNS damage is limited. We report in this study that ssRNA causes neurodegeneration and neuroinflammation dependent on TLR7 in the CNS. TLR7 is not only expressed in microglia, the major immune cells of the brain, but also in neurons of the CNS. Extracellularly delivered ssRNA40, an oligoribonucleotide derived from HIV and an established ligand of TLR7, induces neuronal cell death dependent on TLR7 and the central adapter molecule MyD88 in vitro. Activation of caspase-3 is involved in neuronal damage mediated by TLR7. This cell-autonomous neuronal cell death induced by ssRNA40 is amplified in the presence of microglia that mount an inflammatory response to ssRNA40 through TLR7. Intrathecal administration of ssRNA40 causes widespread neurodegeneration in wild-type but not in TLR7(-/-) mice, confirming that neuronal cell death induced by ssRNA40 through TLR7 occurs in vivo. Our results point to a possible mechanism through which extracellularly delivered ssRNA contributes to CNS damage and determine an obligatory role for TLR7 in this pathway.
The Journal of Immunology 06/2012; 189(3):1448-58. DOI:10.4049/jimmunol.1201078 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-linked protein glycosylation represents an important cellular process for modifying protein properties. It resembles a cascade of various enzymatic reactions, in which class I α-mannosidases play a central role. We and others have recently shown that N-glycosylation plays a major role for immune functions. We now analyzed the expression and function of α-mannosidase I in CD4(+) naive and memory T cells studying human and murine T cells. Alpha-mannosidase I function was altered by (i) treatment with Kifunensine, a specific inhibitor class I α-mannosidases, (ii) synthetic inhibitory RNA, and (iii) overexpression by retroviral gene transfer. T-cell activation was evaluated by CD69 expression, cytokine production and proliferation. Our results demonstrate (i) that α-mannosidase I transcription is transiently downregulated after T-cell activation with either polyclonal anti-CD3/CD28 antibodies or allogeneic CD19(+) B cells, and (ii) that α-mannosidase I exerts an inhibitory effect on T-cell activation. It is interesting to note that the inhibitory effect was restricted to naive CD4(+) T cells in both systems, human T cells and murine transgenic CD4(+)OT-II cells, whereas human memory T cells and primed CD4(+)OT-II cells remained unaffected. Alpha-mannosidase I inhibition reduced the activation threshold for naive but not already primed CD4(+)OT-II cells as the cells were able to respond to lower ovalbumin peptide concentrations and increased the rejection potential of alloreactive T cells in vivo. Thus, complex N-glycans generated by enzymes such as α-mannosidase I inhibit the activation of naive T cells. These findings could be used to improve the ex vivo priming of naive T cells for adaptive T-cell therapies.
[Show abstract][Hide abstract] ABSTRACT: Recent studies demonstrated that primary immune responses can be induced within the brain depending on vessel-associated cells expressing markers of dendritic cells (DC). Using mice transcribing the green fluorescent protein (GFP) under the promoter of the DC marker CD11c, we determined the distribution, phenotype, and source of CD11c+ cells in non-diseased brains. Predilection areas of multiple sclerosis (MS) lesions (periventricular area, adjacent fibre tracts, and optical nerve) were preferentially populated by CD11c+ cells. Most CD11c+ cells were located within the juxtavascular parenchyma rather than the perivascular spaces. Virtually all CD11c+ cells co-expressed ionized calcium-binding adaptor molecule 1 (IBA-1), CD11b, while detectable levels of major histocompatibility complex II (MHC-II) in non-diseased mice was restricted to CD11c+ cells of the choroid plexus. Cellular processes project into the glia limitans which may allow transport and/or presentation of intraparenchymal antigens to extravasated T cells in perivascular spaces. In chimeric mice bearing CD11c-GFP bone marrow, fluorescent cells appeared in the CNS between 8 and 12 weeks after transplantation. In organotypic slice cultures from CD11c-GFP mice, the number of fluorescent cells strongly increased within 72 h. Strikingly, using anti-CD209, an established marker for human DC, a similar population was detected in human brains. Thus, we show for the first time that CD11c+ cells can not only be recruited from the blood into the parenchyma, but also develop from an intraneural precursor in situ. Dysbalance in their recruitment/development may be an initial step in the pathogenesis of chronic (autoimmune) neuroinflammatory diseases such as MS.
[Show abstract][Hide abstract] ABSTRACT: Immune modulating therapies gain increasing importance in treatment of patients with autoimmune diseases such as psoriasis. None of the currently applied biologics achieves significant clinical improvement in all treated patients. Because the therapy with biologics is cost intensive and sometimes associated with side effects, noninvasive diagnostic tools for early prediction of responders are of major interest. We studied the effects of Alefacept (LFA3Ig), an approved drug for treatment of psoriasis, on leukocytes in vitro and in vivo to identify gene markers predictive for treatment response and to further investigate its molecular mechanisms of action. In an open-label study, 20 psoriasis patients were treated weekly with 15 mg Alefacept over 12 wk. We demonstrate that transcription of the tolerance-associated gene (TOAG-1) is significantly up-regulated whereas receptor for hyaluronic acid mediated migration (RHAMM) transcription is down-regulated in PBMCs of responding patients before clinical improvement. TOAG-1 is exclusively localized within mitochondria. Overexpression of TOAG-1 in murine T cells leads to increased susceptibility to apoptosis. Addition of Alefacept to stimulated human T cells in vitro resulted in reduced frequencies of activated CD137(+) cells, increased TOAG-1 but reduced RHAMM expression. This was accompanied by reduced proliferation and enhanced apoptosis. Inhibition of proliferation was dependent on enhanced PDL1 expression of APCs. Thus, peripheral changes of TOAG-1 and RHAMM expression can be used to predict clinical response to Alefacept treatment in psoriasis patients. In the presence of APCs Alefacept can inhibit T cell activation and survival by increasing expression of TOAG-1 on T cells and PDL1 on APCs.
The Journal of Immunology 09/2009; 183(6):4077-87. DOI:10.4049/jimmunol.0804351 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.
[Show abstract][Hide abstract] ABSTRACT: Fibre tract injury evokes recruitment of antigen-presenting- and T cells, but does not cause autoimmune demyelination. This implies that immune tolerance to myelin is actively maintained or readily re-established. Using entorhinal cortex lesion (ECL) to induce axonal degeneration in the hippocampus of adult mice, we studied the induction of B7-H1 (PD-L1) in zones of axonal degeneration. This member of the B7-family has been shown to be expressed on parenchymal cells of various organs, where it strongly down-modulates the activity of T cells. Real-time reverse transcriptase (RT)-PCR revealed low mRNA levels in brain compared to lung and spleen under normal conditions. After ECL, a twofold increase could be observed. Immunocytochemistry revealed astrocytes as source of B7-H1, while immune positive microglia were not detected. Thus, axonal degeneration induces astrocytes to express B7-H1, a potent inhibitor of effector T cells.
[Show abstract][Hide abstract] ABSTRACT: Despite transient, myelin-directed adaptive immune responses in regions of fiber tract degeneration, none of the current models of fiber tract injuries evokes disseminated demyelination, implying effective mechanisms maintaining or re-establishing immune tolerance. In fact, we have recently detected CD95L upregulation accompanied by apoptosis of leukocytes in zones of axonal degeneration induced by entorhinal cortex lesion (ECL), a model of layer-specific axonal degeneration. Moreover, infiltrating monocytes readily transformed into ramified microglia exhibiting a phenotype of immature (CD86+/CD80-) antigen-presenting cells. We now report the appearance of the axonal antigen neurofilament-light along with increased T cell apoptosis and enhanced expression of the pro-apoptotic gene Bad in cervical lymph nodes after ECL. In order to test the functional significance of such local and systemic depletory/regulatory mechanisms on subsequent immunity to central nervous system antigens, experimental autoimmune encephalomyelitis was induced by proteolipid protein immunization 30 days after ECL. In three independent experiments, we found significantly diminished disease scores and infiltrates in lesioned compared to sham-operated SJL mice. This is consistent with a previous meta-statistical analysis (Goodin et al. in Neurology 52:1737-1745, 1999) rejecting the O-hypothesis that brain trauma causes or exacerbates multiple sclerosis. Conversely, brain injuries may involve long-term tolerogenic effects towards brain antigens.
Experimental Brain Research 05/2007; 178(4):542-53. DOI:10.1007/s00221-006-0758-2 · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.
The FASEB Journal 03/2007; 21(2):415-26. DOI:10.1096/fj.06-6130com · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although drainage pathways of soluble antigens from brain to cervical lymph nodes have been well established, there is no direct evidence for similar routes of leukocytes leaving the central nervous system. We developed a protocol allowing the cross-sectioning of an entire head-neck preparation while preserving the signal of the GFP. We monitored how GFP-expressing CD4 T lymphocytes injected into the entorhinal cortex after lesion or the lateral ventricle of unlesioned C57/bl6 mice reach cervical lymph nodes. Irrespective of the injection site, we demonstrate their passage through the cribroid plate, appearance in the nasal mucosa, and specific accumulation in one of the cervical lymph nodes.
[Show abstract][Hide abstract] ABSTRACT: Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.
American Journal of Transplantation 03/2005; 5(2):268-81. DOI:10.1111/j.1600-6143.2005.00689.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clinically, an increasing number of older recipients are listed for transplantation. We examined recipient age-associated alterations of the immune response and their effects on graft function.
Three- and 18-month-old Lewis (LEW) rats received kidneys from 3- and 18-month-old Fischer 344 (F344) rats (1.5 mg/kg/d cyclosporine A for 10 days; n=6/group) and were observed for 180 days. In additional groups, double kidney transplantations were performed to determine the impact of nephron mass and recipient age on graft outcome.
All young recipients but only 66% of old recipients survived the observation period. Increasing recipient age resulted in a significant decrease in renal allograft function (P<0.001), more advanced morphologic evidence of chronic allograft damage (P<0.001), and greater cellular infiltration (P<0.05) and major histocompatibility complex expression (P<0.01) within grafts. Additional in vitro studies examined age-related changes in the cellular immune response by enzyme-linked immunosorbent assay, fluorescence-activated cell sorter analysis, and alloreactive enzyme-linked immunospot: splenocytes from old LEW rats produced significantly more interleukin (IL)-2 (P<0.0001), IL-4 (P<0.05), interferon (IFN)-gamma (P<0.0001), and tumor necrosis factor-alpha (P<0.05). IFN-gamma-producing memory-type T cells were significantly elevated in older rats (P<0.0001). Moreover, they revealed significantly more alloreactive T cells directed against F344 (146 +/- 64.2 and 512 +/- 277/10(6) T cells; P<0.05). Double renal allografts from young donors into old recipients confirmed an independent effect of recipient age on the acceleration of chronic graft deterioration.
The enhanced cellular immune responsiveness in elderly recipients was associated with advanced chronic graft injury. Clinically, older recipients may need a modified immunosuppression.
[Show abstract][Hide abstract] ABSTRACT: The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MHICI) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Ad(beta)-gal adenovirus, encoding for the reporter gene E. coli beta-galactosidase, in the presence of various polybrene concentrations. We determined the amount of beta-gal positive cells by X-gal staining and the beta-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Ad(beta)-gal resulted in a marked increase of beta-gal positive cells and beta-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.
[Show abstract][Hide abstract] ABSTRACT: Viral promoters are commonly used as regulatory elements in gene therapy vectors due to their strong activity in various cell lines in vitro. However, transgene expression under the control of viral promoters in vivo has been shown to be limited to a short period of time. Several mechanisms for the transient expression of the delivered transgene may be important including deletion of transduced cells or promoter downregulation. Recently we reported that cytokines may either decrease or increase the activity of the human cytomegalovirus (hCMV) promoter in monocytes depending on the differentiation status of the transduced cells. For many applications, the gene of interest has to be delivered into an inflammatory milieu (tumour, ischaemia/reperfusion, vector-induced inflammation etc.). In this report we investigated the influence of various inflammatory cytokines on the hCMV-IE promoter activity in transduced human primary endothelial cells (Huvec) in vitro, which may be the first target cells after gene transfer into different organs. Cultured cells were infected with an E1-deleted adenoviral vector encoding for E. coliβ-galactosidase (Adβ-gal) driven by the hCMV-IE promoter and incubated either with or without various cytokines. Our results indicate that interferon-γ (IFN-γ) and interleukin-10 (IL-10) downregulate promoter activity in endothelial cells whereas, in contrast, tumour necrosis factor (TNF-α), interleukin 1β (IL-1β) and interleukin 4 (IL-4) increased the promoter activity. These results suggest that inflammatory processes influence the in vivo expression of transferred viral promoter controlled genes of interest.
[Show abstract][Hide abstract] ABSTRACT: T lymphocytes, regardless of their specificity, are considered key targets for genetic modification in the treatment of inherited or acquired human diseases. In this study, we generated Lewis T cell lines specific for Dark Agouti rat alloantigens and tested the potential of allospecific T lymphocytes as carriers of genes encoding therapeutic proteins in transplantation gene therapy. These allospecific T lymphocytes were successfully, stably transduced with enhanced green fluorescent protein (EGFP) by an Mo-MuLV-based retrovirus vector. A novel gene delivery protocol was utilized, resulting in nearly 100% EGFP-expressing T cells. This approach enabled tracking of allospecific transduced T cells in vivo and illustrates their transgene production by fluorometric determination after ex vivo isolation. Quantitation of EGFP transgene expression was used to determine the influence of T cell receptor-specific activation on transgene regulation. A strict positive correlation between activation state and expression level was detected in vitro and in vivo. The activation-induced increase in transgene expression could be blocked by interference with T cell activation signaling pathways by cyclosporin A, anti-CD4 MAb, or CTLA4-Ig. These data provide strong evidence that direct or indirect effects caused by activation-induced transcription factors are crucial in transgene upregulation. Allospecific activation in spleens, lymph nodes, and transplanted grafts can be considered as antigen-specific targeting strategy. This activation might be useful in expressing therapeutic proteins such as TGF-beta or IL-10 specific to these sites. T lymphocyte priming and activation might be prevented or altered by modification of the local microenvironments, thereby exerting a therapeutic influence on acute and chronic graft rejection processes.
Human Gene Therapy 07/2000; 11(9):1303-11. DOI:10.1089/10430340050032401 · 3.76 Impact Factor