CD8.sup.+ T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8.sup.+ T cells recognize cells expressing target antigens, become activated, and secrete IFNgamma. ...However, there are no methods to recognize individual cells that respond to IFNgamma. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNgamma. To identify individual mouse brain cells that respond to IFNgamma we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNgamma signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNgamma to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8.sup.+ T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNgamma. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNgamma by anti-viral CD8.sup.+ T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses.
Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma ...(glioblastoma multiforme GBM) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of
in vivo
phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.
Issue Title: Treatment of Sleep Disorders Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively ...elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B NF-κB inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the clinic.PUBLICATION ABSTRACT
Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus ...failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain. We tested the prediction that expression of fms-like tyrosine kinase ligand 3 (Flt3L) in the brain will recruit dendritic cells and induce a systemic immune response against exogenous influenza hemagglutinin in BALB/c mice. Coexpression of Flt3L with HA in the brain parenchyma induced a robust systemic anti-HA immune response, and a small response against myelin basic protein and proteolipid protein epitopes. Depletion of CD4⁺CD25+ regulatory T cells (Tregs) enhanced both responses. To investigate the autoimmune impact of these immune responses, we characterized the neuropathological and behavioral consequences of intraparenchymal injections of Flt3L and HA in BALB/c and C57BL/6 mice. T cell infiltration in the forebrain was time and strain dependent, and increased in animals treated with Flt3L and depleted of Tregs; however, we failed to detect widespread defects in myelination throughout the forebrain or spinal cord. Results of behavioral tests were all normal. These results demonstrate that Flt3L overcomes the brain's immune privilege, and supports the clinical development of Flt3L as an adjuvant to stimulate clinically effective immune responses against brain neo-antigens, for example, those associated with brain tumors.
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In vitro, IFN‐γ secretion is focused towards the immunological synapse (IS), while secretion of TNF‐α is multi‐directional. We have previously demonstrated the polarization of IFN‐ γ ...within T cells establishing Kupfer‐type or non‐Kupfer type IS with virally infected astrocytes in vivo. We developed a novel adenoviral vector system containing an IFN‐ γreporter to identify IFNγ secretion at immunological synapse in vivo. Astrocytes were infected with this adenovirus, and 30 days later we induced a systemic immune response against Ads, and 14 days after the immunization animals were sacrificed and brains studied by immunohistochemistry and confocal microscopy. Brain sections were stained for TCR, LFA‐1, TK, and IFN‐ γ. Confocal analysis confirmed IFN‐γ polarization, and quantitative analysis was used to demonstrate the expression from the IFNγ ‐reporter adenovirus in astrocytes of immunized mice in contact with CTLs, when compared to non‐immunized mice. These data suggests that IFN‐γ is secreted across the immunological synapse in vivo towards the target virally infected cell.
This work was funded by grants from NINDS, NIH.
We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the ...immune-stimulatory cytokine, i.e.,
fms
-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6
-/-
mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6
-/-
mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of
Prdm 1
(encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.
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Immunological synapses (IS) mediate intercellular communication between T cells and APCs. T cell polarization at the Kupfer‐type IS consists of a peripheral supramolecular activation ...cluster (pSMAC), which is high in LFA‐1, and a central SMAC, high in TCR. Target virally infected astrocytes also polarize towards the immunological synapse
in vivo
. Astrocyte polarization in response to physical lesions
in vitro
is mediated through the activation of a signaling pathway mediated by a family of Rho‐GTPases, and coordinated by Cdc42. Here we tested the hypothesis that that stimulation of a Rho‐GTPase cascade underlies astrocyte polarization in response to an immune attack
in vitro
and
in vivo
. To do so, we infected brain astrocytes with a novel adenoviral (Ad) vector expressing a dominant negative variant of Cdc42 (Cdc42DN) and Thymidine Kinase (TK), a marker of infected cells, and analyzed their responses to a T cell attack. We show that during an anti‐viral immune response, infected astrocytes contacted by CTLs become polarized through the activation of a Rho‐GTPase signaling pathway.
This work was funded by grants from NINDS/NIH.
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We studied the ability of antigen presenting cells (APC) infiltrating intracranial GBM to phagocytose and transport tumor antigens after combined intratumoral delivery of Ads encoding ...the conditional cytotoxic, thymidine kinase (TK) and the immune‐stimulant, Flt3L. We investigated whether the immune cells that infiltrate intracranial GBM internalize fluorescent FluoSpheres implanted intratumorally 4 days after Ad‐Flt3L/Ad‐TK treatment. Distribution of FluoSpheres was widespread throughout the tumor mass, allowing wide availability for infiltrating immune cells. Beads were rapidly taken up 24 h after administration by intratumoral macrophages and dendritic cells (DCs). APC containing beads were detected within the tumor up to 7 days after bead delivery approaching or entering tumor blood vessels. Four days after bead delivery, bead‐containing APCs were detected in the draining cervical lymph nodes. Our results show that APC infiltrating intracranial GBM are capable of antigen uptake and migration from the brain to the draining lymph node after Ad‐TK/GCV+Ad‐Flt3L treatment where they would present GBM antigen to T cells. Support: NIH/NINDS R01 NS44556.01, R21‐NSO54143.01; UO1 NS052465.01; RO3 TW006273‐01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309‐01, and R21 NS047298‐01 to P.R.L; F32 NS058156.01 to M.C.
Immune privilege, tumor immune evasion and a lack of dendritic cells (DC) in the normal brain parenchyma all contribute to immunological ignorance against glioblastoma multiforme (GBM) antigen. Here ...we uncovered a novel pathway for the activation of GBM antigen‐specific T‐cell dependent immune response which is mediated by the release of the high‐mobility‐group box 1 (HMGB1) by dying tumor cells after treatment with TK (+GCV). Endogenous TLR2 signaling induces infiltration of bone marrow derived DC into GBM after the intratumoral expression of Flt3L and TK. In the absence of TLR2 signaling, tumor infiltrating DC fail to stimulate the proliferation and activation of tumor antigen specific T cells and CD8+ T cell dependent tumor regression is ablated. Blocking HMGB1 activity in vivo using glycyrrhizin or HMGB1 depleting antibodies inhibited Flt3L/TK induced brain tumor regression. Thus, endogenous TLR2 signaling mediated via HMGB1 plays a critical role in promoting a CD8+ T cell dependent anti‐GBM immune response and could be exploited as a novel adjuvant therapy to overcome immunological privilege in the brain.
Supported by NIH/NINDS: 1R01 NS44556.01, NS445561.01; 1R21‐NSO54143.01; 1UO1 NS052465.01, 1 RO3 TW006273‐01 to MGC. NIH/NINDS: 1 RO1 NS 054193.01; RO1 NS 42893.01, U54 NS045309‐01 and 1R21 NS047298‐01 to PRL. NIH/NINDS 1F32 NS058156.01 to MC.
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