Klotho deficiency affects the spine morphology and network synchronization of neurons.

Klotho-deficient mice rapidly develop cognitive impairment and show some evidence of the onset of neurodegeneration. However, it is impossible to investigate the long-term consequences on the brain because of the dramatic shortening of lifespan caused by systemic klotho deficiency. As klotho expression is downregulated with advancing organismal age, understanding the mechanisms of klotho action is important for developing novel strategies to support healthy brain aging. Previously, we reported that klotho-deficient mice show enhanced long-term potentiation prior to the onset of cognitive impairment. To inform this unusual phenotype, herein, we examined neuronal structure and in vitro synaptic function. Our results indicate that klotho deficiency causes the population of dendritic spines to shift towards increased head diameter and decreased length consistent with mature, mushroom type spines. Multi-electrode array recordings from klotho-deficient neurons show increased synchronous firing and activity changes reflective of increased neuronal network activity. Supplementation of the neuronal growth media with recombinant shed klotho corrected some but not all of the activity changes caused by klotho deficiency. Last, in vivo we found that klotho-deficient mice have a decreased latency to induced seizure activity. Together these data show that klotho-deficient memory impairments are underpinned by structural and functional changes that may preclude ongoing normal cognition.

Role of fibroblast growth factor 23 and klotho cross talk in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia that mainly affects the elderly. Several reports have demonstrated that aging is involved in the underlying pathogenic mechanisms of IPF. α-Klotho (KL) has been well characterized as an "age-suppressing" hormone and can provide protection against cellular senescence and oxidative stress. In this study, KL levels were assessed in human plasma and primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF-FB) and in lung tissue from mice exposed to bleomycin, which showed significant downregulation when compared with controls. Conversely, transgenic mice overexpressing KL were protected against bleomycin-induced lung fibrosis. Treatment of human lung fibroblasts with recombinant KL alone was not sufficient to inhibit transforming growth factor-ß (TGF-ß)-induced collagen deposition and inflammatory marker expression. Interestingly, fibroblast growth factor 23 (FGF23), a proinflammatory circulating protein for which KL is a coreceptor, was upregulated in IPF and bleomycin lungs. To our surprise, FGF23 and KL coadministration led to a significant reduction in fibrosis and inflammation in IPF-FB; FGF23 administration alone or in combination with KL stimulated KL upregulation. We conclude that in IPF downregulation of KL may contribute to fibrosis and inflammation and FGF23 may act as a compensatory antifibrotic and anti-inflammatory mediator via inhibition of TGF-ß signaling. Upon restoration of KL levels, the combination of FGF23 and KL leads to resolution of inflammation and fibrosis. Altogether, these data provide novel insight into the FGF23/KL axis and its antifibrotic/anti-inflammatory properties, which opens new avenues for potential therapies in aging-related diseases like IPF.

Cardiovascular Effects of Renal Distal Tubule Deletion of the FGF Receptor 1 Gene.

The bone-derived hormone fibroblast growth factor-23 (FGF-23) activates complexes composed of FGF receptors (FGFRs), including FGFR1, and α-Klotho in the kidney distal tubule (DT), leading to increased sodium retention and hypertension. However, the role of FGFR1 in regulating renal processes linked to hypertension is unclear. Here, we investigated the effects of selective FGFR1 loss in the DT. Conditional knockout (cKO) of FGFR1 in the DT (FGFR1DT-cKO mice) resulted in left ventricular hypertrophy (LVH) and decreased kidney expression of α-Klotho in association with enhanced BP, decreased expression of angiotensin converting enzyme 2, and increased expression of the Na+-K+-2Cl- cotransporter. Notably, recombinant FGF-23 administration similarly decreased the kidney expression of α-Klotho and induced LVH in mice. Pharmacologic activation of FGFR1 with a monoclonal anti-FGFR1 antibody (R1MAb1) normalized BP and significantly attenuated LVH in the Hyp mouse model of excess FGF-23, but did not induce a response in FGFR1DT-cKO mice. The hearts of FGFR1DT-cKO mice showed increased expression of the transient receptor potential cation channel, subfamily C, member 6 (TRPC6), consistent with cardiac effects of soluble Klotho deficiency. Moreover, administration of recombinant soluble Klotho lowered BP in the Hyp mice. Thus, FGFR1 in the DT regulates systemic hemodynamic responses opposite to those predicted by the actions of FGF-23. These cardiovascular effects appear to be mediated by paracrine FGF control of kidney FGFR1 and subsequent regulation of soluble Klotho and TRPC6. FGFR1 in the kidney may provide a new molecular target for treating hypertension.

Fibroblast growth factor 23 and Klotho contribute to airway inflammation.

Circulating levels of fibroblast growth factor (FGF)23 are associated with systemic inflammation and increased mortality in chronic kidney disease. α-Klotho, a co-receptor for FGF23, is downregulated in chronic obstructive pulmonary disease (COPD). However, whether FGF23 and Klotho-mediated FGF receptor (FGFR) activation delineates a pathophysiological mechanism in COPD remains unclear. We hypothesised that FGF23 can potentiate airway inflammation via Klotho-independent FGFR4 activation.FGF23 and its effect were studied using plasma and transbronchial biopsies from COPD and control patients, and primary human bronchial epithelial cells isolated from COPD patients as well as a murine COPD model.Plasma FGF23 levels were significantly elevated in COPD patients. Exposure of airway epithelial cells to cigarette smoke and FGF23 led to a significant increase in interleukin-1ß release via Klotho-independent FGFR4-mediated activation of phospholipase Cγ/nuclear factor of activated T-cells signalling. In addition, Klotho knockout mice developed COPD and showed airway inflammation and elevated FGFR4 expression in their lungs, whereas overexpression of Klotho led to an attenuation of airway inflammation.Cigarette smoke induces airway inflammation by downregulation of Klotho and activation of FGFR4 in the airway epithelium in COPD. Inhibition of FGF23 or FGFR4 might serve as a novel anti-inflammatory strategy in COPD.

Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

Biochemical and functional characterization of the klotho-VS polymorphism implicated in aging and disease risk.

Klotho (KL) is an age-regulating protein named after the Greek goddess who spins the thread of life. Mice deficient in KL are normal throughout development, but rapidly degenerate and display a variety of aging-associated abnormalities that eventually lead to decreased life expectancy. While multiple genetic association studies have identified KL polymorphisms linked with changes in disease risk, there is a paucity of concrete mechanistic data to explain how these amino acid substitutions alter KL protein function. The KLVS polymorphism is suggested to lead to changes in protein trafficking although the mechanism is unclear. Our studies have sought to further investigate the functional differences in the KLVS variant that result in increased risk of many age-related diseases. Our findings suggest that the F352V and C370S substitutions lead to alterations in processing as seen by differences in shedding and half-life. Their co-expression in KLVS results in a phenotype resembling wild-type, but despite this intragenic complementation there are still changes in homodimerization and interactions with FGFR1c. Taken together, these studies suggest that KLVS leads to altered homodimerization that indirectly leads to changes in processing and FGFR1c interactions. These findings help elucidate the functional differences that result from the VS polymorphism, which will help clarify how alterations in KL function can lead to human disease and affect cognition and lifespan.

Identification of novel small molecules that elevate Klotho expression.

The absence of Klotho (KL) from mice causes the development of disorders associated with human aging and decreased longevity, whereas increased expression prolongs lifespan. With age, KL protein levels decrease, and keeping levels consistent may promote healthier aging and be disease-modifying. Using the KL promoter to drive expression of luciferase, we conducted a high-throughput screen to identify compounds that activate KL transcription. Hits were identified as compounds that elevated luciferase expression at least 30%. Following validation for dose-dependent activation and lack of cytotoxicity, hit compounds were evaluated further in vitro by incubation with opossum kidney and Z310 rat choroid plexus cells, which express KL endogenously. All compounds elevated KL protein compared with control. To determine whether increased protein resulted in an in vitro functional change, we assayed FGF23 (fibroblast growth factor 23) signalling. Compounds G-I augmented ERK (extracellular-signal-regulated kinase) phosphorylation in FGFR (fibroblast growth factor receptor)-transfected cells, whereas co-transfection with KL siRNA (small interfering RNA) blocked the effect. These compounds will be useful tools to allow insight into the mechanisms of KL regulation. Further optimization will provide pharmacological tools for in vivo studies of KL.

Circadian regulation of dentate gyrus excitability mediated by G-protein signaling.

The central circadian regulator within the suprachiasmatic nucleus transmits time of day information by a diurnal spiking rhythm driven by molecular clock genes controlling membrane excitability. Most brain regions, including the hippocampus, harbor similar intrinsic circadian transcriptional machinery, but whether these molecular programs generate oscillations of membrane properties is unclear. Here, we show that intrinsic excitability of mouse dentate granule neurons exhibits a 24-h oscillation that controls spiking probability. Diurnal changes in excitability are mediated by antiphase G-protein regulation of potassium and sodium currents that reduce excitability during the Light phase. Disruption of the circadian transcriptional machinery by conditional deletion of Bmal1 enhances excitability selectively during the Light phase by removing G-protein regulation. These results reveal that circadian transcriptional machinery regulates intrinsic excitability by coordinated regulation of ion channels by G-protein signaling, highlighting a potential novel mechanism of cell-autonomous oscillations.

In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain.

The microanatomy of immune clearance of infected brain cells remains poorly understood. Immunological synapses are essential anatomical structures that channel information exchanges between T cell-antigen-presenting cells (APC) during the priming and effector phases of T cells' function, and during natural killer-target cell interactions. The hallmark of immunological synapses established by T cells is the formation of the supramolecular activation clusters (SMACs), in which adhesion molecules such as leukocyte function-associated antigen 1 segregate to the peripheral domain of the immunological synapse (p-SMAC), which surrounds the T cell receptor-rich or central SMAC (c-SMAC). The inability so far to detect SMAC formation in vivo has cast doubts on its functional relevance. Herein, we demonstrate that the in vivo formation of SMAC at immunological synapses between effector CD8+ T cells and target cells precedes and mediates clearance of virally infected brain astrocytes.

Combined Flt3L/TK gene therapy induces immunological surveillance which mediates an immune response against a surrogate brain tumor neoantigen.

Glioblastoma multiforme (GBM) is a primary brain tumor with a median survival of 14.6 months postdiagnosis. The infiltrative nature of GBM prevents complete resection and residual brain tumor cells give rise to recurrent GBM, a hallmark of this disease. Recurrent GBMs are known to harbor numerous mutations/gene rearrangements when compared to the primary tumor, which leads to the potential expression of novel proteins that could serve as tumor neoantigens. We have developed a combined immune-based gene therapeutic approach for GBM using adenoviral (Ads) mediated gene delivery of Herpes Simplex Virus Type 1-thymidine kinase (TK) into the tumor mass to induce tumor cells' death combined with an adenovirus expressing fms-like tyrosine kinase 3 ligand (Flt3L) to recruit dendritic cells (DCs) into the tumor microenvironment. This leads to the induction of specific anti-brain tumor immunity and immunological memory. In a model of GBM recurrence, we demonstrate that Flt3L/TK mediated immunological memory is capable of recognizing brain tumor neoantigens absent from the original treated tumor. These data demonstrate that the Flt3L/TK gene therapeutic approach can induce systemic immunological memory capable of recognizing a brain tumor neoantigen in a model of recurrent GBM.

A chromatin modulator sustains self-renewal and enables differentiation of postnatal neural stem and progenitor cells.

It remains unknown whether H3K4 methylation, an epigenetic modification associated with gene activation, regulates fate determination of the postnatal neural stem and progenitor cells (NSPCs). By inactivating the Dpy30 subunit of the major H3K4 methyltransferase complexes in specific regions of mouse brain, we demonstrate a crucial role of efficient H3K4 methylation in maintaining both the self-renewal and differentiation capacity of postnatal NSPCs. Dpy30 deficiency disrupts development of hippocampus and especially the dentate gyrus and subventricular zone, the major regions for postnatal NSC activities. Dpy30 is indispensable for sustaining the self-renewal and proliferation of NSPCs in a cell-intrinsic manner and also enables the differentiation of mouse and human neural progenitor cells to neuronal and glial lineages. Dpy30 directly regulates H3K4 methylation and the induction of several genes critical in neurogenesis. These findings link a prominent epigenetic mechanism of gene expression to the fundamental properties of NSPCs and may have implications in neurodevelopmental disorders.

High-capacity adenovirus vector-mediated anti-glioma gene therapy in the presence of systemic antiadenovirus immunity.

Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.

Flt3L in combination with HSV1-TK-mediated gene therapy reverses brain tumor-induced behavioral deficits.

Glioblastoma multiforme (GBM) is an invasive and aggressive primary brain tumor which is associated with a dismal prognosis. We have earlier developed a macroscopic, intracranial, syngeneic GBM model, in which treatment with adenoviral vectors (Ads) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) plus ganciclovir (GCV) resulted in survival of approximately 20% of the animals. In this model, treatment with Ads expressing Fms-like tyrosine kinase 3 ligand (Flt3L), in combination with Ad-HSV1-TK improves the survival rate to approximately 70% and induces systemic antitumor immunity. We hypothesized that the growth of a large intracranial tumor mass would cause behavioral abnormalities that can be reversed by the combined gene therapy. We assessed the behavior and neuropathology of tumor-bearing animals treated with the combined gene therapy, 3 days after treatment, in long-term survivors, and in a recurrent model of glioma. We demonstrate that the intracranial GBM induces behavioral deficits that are resolved after treatment with Ad-Flt3L/Ad-TK (+GCV). Neuropathological analysis of long-term survivors revealed an overall recovery of normal brain architecture. The lack of long-term behavioral deficits and limited neuropathological abnormalities demonstrate the efficacy and safety of the combined Ad-Flt3L/Ad-TK gene therapy for GBM. These findings can serve to underpin further developments of this therapeutic modality, leading toward implementation of a Phase I clinical trial.

Flt3L in Combination With HSV1-TK-mediated Gene Therapy Reverses Brain Tumor-induced Behavioral Deficits.

Glioblastoma multiforme (GBM) is an invasive and aggressive primary brain tumor which is associated with a dismal prognosis. We have earlier developed a macroscopic, intracranial, syngeneic GBM model, in which treatment with adenoviral vectors (Ads) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) plus ganciclovir (GCV) resulted in survival of ∼20% of the animals. In this model, treatment with Ads expressing Fms-like tyrosine kinase 3 ligand (Flt3L), in combination with Ad-HSV1-TK improves the survival rate to ∼70% and induces systemic antitumor immunity. We hypothesized that the growth of a large intracranial tumor mass would cause behavioral abnormalities that can be reversed by the combined gene therapy. We assessed the behavior and neuropathology of tumor-bearing animals treated with the combined gene therapy, 3 days after treatment, in long-term survivors, and in a recurrent model of glioma. We demonstrate that the intracranial GBM induces behavioral deficits that are resolved after treatment with Ad-Flt3L/Ad-TK (+GCV). Neuropathological analysis of long-term survivors revealed an overall recovery of normal brain architecture. The lack of long-term behavioral deficits and limited neuropathological abnormalities demonstrate the efficacy and safety of the combined Ad-Flt3L/Ad-TK gene therapy for GBM. These findings can serve to underpin further developments of this therapeutic modality, leading toward implementation of a Phase I clinical trial.

FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model.

Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone-associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.

FGF-23 Deficiency Impairs Hippocampal-Dependent Cognitive Function.

Fibroblast growth factor receptor (FGFR) and α-Klotho transduce FGF-23 signaling in renal tubules to maintain systemic phosphate/vitamin D homeostasis. Mice deficient for either the ligand, FGF-23, or the co-receptor, Klotho, are phenocopies with both showing rapid and premature development of multiple aging-like abnormalities. Such similarity in phenotype, suggests that FGF-23 and Klotho have co-dependent systemic functions. Recent reports revealed inverse central nervous system (CNS) effects of Klotho deficiency or Klotho overexpression on hippocampal synaptic, neurogenic, and cognitive functions. However, it is unknown whether FGF-23 deficiency effects function of the hippocampus. We report that, similar to Klotho-deficient mice, FGF-23-deficient mice develop dose-dependent, hippocampal-dependent cognitive impairment. However, FGF-23-deficient brains had no gross structural or developmental defects, no change in hippocampal synaptic plasticity, and only minor impairment to postnatal hippocampal neurogenesis. Together, these data provide evidence that FGF-23 deficiency impairs hippocampal-dependent cognition but otherwise results in a brain phenotype that is distinct from the KL-deficient mouse.

The Effects of the Anti-aging Protein Klotho on Mucociliary Clearance.

α-klotho (KL) is an anti-aging protein and has been shown to exert anti-inflammatory and anti-oxidative effects in the lung and pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. The current study investigated the direct effect of KL on the bronchial epithelium in regards to mucociliary clearance parameters. Primary human bronchial and murine tracheal epithelial cells, cultured, and differentiated at the air liquid interface (ALI), were treated with recombinant KL or infected with a lentiviral vector expressing KL. Airway surface liquid (ASL) volume, airway ion channel activities, and expression levels were analyzed. These experiments were paired with ex vivo analyses of mucociliary clearance in murine tracheas from klotho deficient mice and their wild type littermates. Our results showed that klotho deficiency led to impaired mucociliary clearance with a reduction in ASL volume in vitro and ex vivo. Overexpression or exogenous KL increased ASL volume, which was paralleled by increased activation of the large-conductance, Ca2+-activated, voltage-dependent potassium channel (BK) without effect on the cystic fibrosis transmembrane conductance regulator (CFTR). Furthermore, KL overexpression downregulated IL-8 levels and attenuated TGF-ß-mediated downregulation of LRRC26, the γ subunit of BK, necessary for its function in non-excitable cells. In summary, we show that KL regulates mucociliary function by increasing ASL volume in the airways possibly due to underlying BK activation. The KL mediated BK channel activation may be a potentially important target to design therapeutic strategies in inflammatory airway diseases when ASL volume is decreased.

Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model.

The disseminated characteristics of human glioblastoma multiforme (GBM) make it a particularly difficult tumor to treat with long-term efficacy. Most preclinical models of GBM involve treatment of a single tumor mass. For therapeutic outcomes to translate from the preclinical to the clinical setting, induction of an antitumor response capable of eliminating multifocal disease is essential. We tested the hypothesis that expression of Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) and TK (herpes simplex virus type 1-thymidine kinase) within brain gliomas would mediate regression of the primary, treated tumor mass and a secondary, untreated tumor growing at a distant site from the primary tumor and the site of therapeutic vector injection. In both the single-GBM and multifocal-GBM models used, all saline-treated control animals succumbed to tumors by day 22. Around 70% of the animals bearing a single GBM mass treated with an adenovirus expressing Flt3L (AdFlt3L) and an adenovirus expressing TK (AdTK + GCV) survived long term. Approximately 50% of animals bearing a large primary GBM that were implanted with a second GBM in the contralateral hemisphere at the same time the primary tumors were being treated with AdFlt3L and AdTK also survived long term. A second multifocal GBM model, in which bilateral GBMs were implanted simultaneously and only the right tumor mass was treated with AdFlt3L and AdTK, also demonstrated long-term survival. While no significant difference in survival was found between unifocal and multifocal GBM-bearing animals treated with AdFlt3L and AdTK, both treatments were statistically different from the saline-treated control group (p < 0.05). Our results demonstrate that combination therapy with AdFlt3L and AdTK can eradicate multifocal brain tumor disease in a syngeneic, intracranial GBM model.

Klotho, the Key to Healthy Brain Aging?

Brain expression of klotho was first described with the initial discovery of the klotho gene. The prominent age-regulating effects of klotho are attributed to regulation of ion homeostasis through klotho function in the kidney. However, recent advances identified brain functions and cell populations, including adult hippocampal neural progenitors, which require klotho. As well, both human correlational studies and mouse models of disease show that klotho is protective against multiple neurological and psychological disorders. This review focuses on current knowledge as to how the klotho protein effects the brain.

Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model.

In spite of preclinical efficacy and recent randomized, controlled studies with adenoviral vectors expressing herpes simplex virus-1 thymidine kinase (HSV1-TK) showing statistically significant increases in survival, most clinical trials using single therapies have failed to provide major therapeutic breakthroughs. Because glioma is a disease with dismal prognosis and rapid progression, it is an attractive target for gene therapy. Preclinical models using microscopic brain tumor models (e.g., < or =0.3 mm3) may not reflect the pathophysiology and progression of large human tumors. To overcome some of these limitations, we developed a syngeneic large brain tumor model. In this model, administration of single therapeutic modalities, either conditional cytotoxicity or immunostimulation, fail. However, when various immunostimulatory therapies were delivered in combination with conditional cytotoxicity (HSV1-TK), only the combined delivery of fms-like tyrosine kinase ligand (Flt3L) and HSV1-TK significantly prolonged the survival of large tumor-bearing animals (> or =80%; P < or = 0.005). When either macrophages or CD4+ cells were depleted before administration of viral therapy, TK + Flt3L therapy failed to prolong survival. Meanwhile, depletion of CD8+ cells or natural killer cells did not affect TK + Flt3L efficacy. Spinal cord of animals surviving 6 months after TK + Flt3L were evaluated for the presence of autoimmune lesions. Whereas macrophages were present within the corticospinal tract and low levels of T-cell infiltration were detected, these effects are not indicative of an overt autoimmune disorder. We propose that combined Flt3L and HSV1-TK adenoviral-mediated gene therapy may provide an effective antiglioma treatment with increased efficacy in clinical trials of glioma.