Transposon-mediated Gliomagenesis to Investigate Dendritic Cell Trafficking after Immunotherapy in a Tolerized Host

John R. Ohlfest, Ph.D.

University of Minnesota

Funded in June, 2008: $200000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY .

LAY SUMMARY

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Imaging May Reveal Whether a Specific Type of Immune Cell is Key to Effective Brain Tumor Treatment

The investigators will use cellular imaging techniques in a new animal model of lethal brain tumor to determine whether increasing the quantities of a specific type of immune cell in the brain is associated with improved therapeutic outcomes.

Glioblastoma is a fatal primary brain tumor.  The tumor cells migrate throughout the brain, rendering surgical removal or radiation incapable of eradicating them. A promising approach, therefore, is using “immunotherapies,” therapeutic vaccines that stimulate immune cells to enter the brain and attack the tumor. Efforts to determine which types of immune cells may have the most potential to kill off brain tumor cells, however, have been hampered by lack of an animal model that closely resembles human glioma. Recently, a mouse model that is similar to human glioma was developed by University of Minnesota investigators.  As in humans, the immune cells in this mouse model are initially “tolerized” to the tumor, meaning that they fail to recognize that the tumor cells are foreign and therefore do not attack them. But when the animal receives a therapeutic vaccine, the vaccine provokes an immune response.

The researchers hypothesize that a specific type of immune cell, called “a Natural Killer dendritic cell,” identifies tumor cells and attacks the tumor cells directly, while also summoning immune T cells into the brain to reinforce the attack. In fact, they hypothesize, the quantity of natural killer dendritic cells that enters the brain will be directly correlated with the extent of treatment benefits. They have developed cellular imaging techniques to determine if their hypothesis is correct.  In the mice that are treated with a therapeutic vaccine, they will use double two-photon imaging with bioluminescence to simultaneously visualize tumor cells (which will glow blue) and natural killer dendritic cells (which will glow red). They will measure whether increased quantities of natural killer dendritic cells in the brain correlate with reduced quantities of brain tumor cells.  If so, they will confirm these results through use of a related imaging technique called quantum dot imaging, which can be undertaken in glioma patients in the foreseeable future. Ultimately, the research may lead to improved therapies that stimulate greater quantities of these natural killer dendritic cells to enter the brain and kill off glioma cells.

Significance:  The imaging research may determine whether immune system natural killer dendritic cells are potentially potent brain tumor fighters, and lead the use of imaging in giloma patients to assess how effectively various immunotherapies strengthen this immune response.

ABSTRACT

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Transposon-mediated Gliomagenesis to Investigate Dendritic Cell Trafficking after Immunotherapy in a Tolerized Host

Dendritic cells (DC) are sentinels of the innate immune system that play an essential role in educating T lymphocytes how to respond to pathogen and tumor antigens. DC vaccines have induced cytotoxic T lymphocyte (CTL) responses against tumors experimentally and in human cancer patients.  The rationale for using immunotherapy against glioblastoma multiforme (GBM), a destructive and lethal brain tumor, is the “seek and destroy” capacity of CTLs that can infiltrate the brain to kill tumor left behind after surgery. Tumor infiltrating T cells have been detected in GBM patients treated by DC vaccines, and extensively characterized by histological and molecular assays. Despite these encouraging results, immunotherapy is typically not curative for GBM patients. 

The concentration of tumor infiltrating DC (TIDC) is positive prognostic factor for many cancers, yet their significance in brain tumors is poorly understood. Moreover, almost nothing is known about the temporal dynamics of DC trafficking into brain tumors, how that changes during immunotherapy, and the responsible mechanisms for inducing DC trafficking. Mounting evidence suggests that DC not only regulate anti-tumor T cell responses, but that some DC subsets can actually kill tumor directly.  The central hypothesis of this grant is that the quantity of specific subsets of glioma-infiltrating DC significantly impacts the outcome of immunotherapy.  This is a new hypothesis that may also be controversial.  As such, testing this hypothesis will require unique animal models and in vivo imaging technologies in order to provide adequate supporting data.

EGFRvIII is a truncated, hyperactive version of the epidermal growth factor receptor (EGFR) that represents one of the few “glioma-specific” antigens expressed by tumor cells but not on normal tissue. We and others have targeted EGFRvIII using immunotherapy, and clinical trials are ongoing. Progress in this area of immunotherapy has been hindered by lack of a tractable animal model in which the tumor expresses the relevant antigen (e.g., EGFRvIII) but the host is tolerized to the antigen (as in human patients). We have recently developed a novel mouse model of glioblastoma that expresses human antigens using somatic cell gene transfer for evaluation of DC trafficking and immunotherapy.

The development of this glioma model is significant because it arises spontaneously and is more relevant than cell line-induced models that have failed to predict clinical outcomes.  In this model, gliomas are induced that express renilla luciferase, and DCs can be adoptively transferred from firefly luciferase donors, thereby allowing both tumor and immune cells to be tracked by two-signal bioluminescent imaging in vivo.  In Specific Aim 1, the trafficking of DC into glioma will be determined using two-signal bioluminescent imaging in mice treated by EGFRvIII vaccination. In addition, the trafficking of DC from the tumor into the draining lymph nodes will be determined and correlated to survival to define a quantitative relationship between trafficking and survival. These trafficking experiments will be corroborated by conducting a detailed analysis of TIDC using histology and flow cytometry. In a second set of experiments, chemokines IP-10, CCL21 and Flt3L will be overexpressed in the glioma using regulatable gene transfer technology.   The conclusive role for these chemokines in DC trafficking will be shown using antibody-mediated interference.

The dogma in the DC field has been that “conventional” myeloid DC (mDC) are most important for priming anti-tumor immunity, and either T cells or natural killer (NK)  cells are the effectors that directly kill tumor. Very recently, a novel subset of DC has been identified that is able to directly kill tumor cells, secrete high levels of interferon gamma (IFN-gamma), and also present antigen to naïve T cells.  This novel subset of DC has been named “natural killer dendritic cells” (NKDCs). Several high-impact papers have demonstrated that NKDCs produce IFN-gamma and directly kill tumor to a greater extent than conventional NK cells.  If these results are true, it significantly alters our understanding on the influence of DC in anti-tumor immunity. However, absolutely nothing is known about the behavior of NKDCs in the brain, or about their potential as a novel cellular therapy for glioma. 

In Specific Aim 2, we will induce tumors in T cell-deficient mice, then adoptively transfer luciferase-labeled NKDC directly into the glioma and determine direct anti-tumor activity by bioluminescent imaging and survival. This experiment will also be done in immunocompetent mice to investigate if NKDC can prime adaptive anti-tumor immunity.  While two-signal bioluminescent imaging is a powerful tool, this imaging may be difficult to translate to human studies.  Therefore, we will also label NKDC with fluorescent quantum dots (Qdot).  This approach has recently been used to locate the draining lymph nodes in large animals by in vivo imaging.  Qdot-labeled NKDC will be infused directly into the tumor or intravenously.  Trafficking will be measured by bioluminescent imaging as positive control, but then overlayed with Qdot fluorescence to detect draining lymph node involvement.  The therapeutic effects of NKDC will be determined by measuring tumor growth and survival of tumor-bearing mice.   Collectively, these experiments will provide new insights into the trafficking and anti-glioma activity of conventional DC and NKDC, with an eye on human clinical applications in the foreseeable future.

INVESTIGATOR BIOGRAPHIES

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John R. Ohlfest, Ph.D.

Dr. Ohlfest is an Assistant Professor of Pediatrics and Neurosurgery and Director of Neurosurgical Gene Therapy at the University of Minnesota.  He received a Bachelor of Science degree in biology from Iowa State University, and a Ph.D. in genetics and molecular biology from the University of Minnesota.  Dr. Ohlfest’s laboratory focuses on improving immunotherapy for treatment of brain tumors.  They have discovered a new method to study brain tumors that spontaneously arise in mice  in which the tumor cells emit light that indicates tumor growth.  Using this model, they developed a vaccine that stimulates the body’s own immune cells (T cells) to attack and kill brain tumor cells.  Recently they discovered that another immune cell known as a Natural Killer cell may play central role in regulating (and improving) the ability of the T cells to recognize and kill tumor cells.  In fact it was shown the Natural Killer cells can infiltrate the brain and assist the T cells in a coordinated assault on the tumor.  Their current research aims to further define the mechanisms that influence immune-mediated brain tumor cell killing and translate these findings into clinical trials for brain tumor therapy.