Aaron J. Johnson

Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors

Treatment of advanced, poorly immunogenic tumors in animal models, considered the closest simulation available thus far for conditions observed in cancer patients, remains a major challenge for cancer immunotherapy. We reported previously that established tumors in mice receiving an agonistic mAb to the T cell costimulatory molecule 4-1BB (CD137) regress due to enhanced tumor antigen-specific cytotoxic T lymphocyte responses. In this study, we demonstrate that several poorly immunogenic tumors, including C3 tumor, TC-1 lung carcinoma, and B16-F10 melanoma, once established as solid tumors or metastases, are refractory to treatment by anti-4-1BB mAb. We provide evidence that immunological ignorance, rather than anergy or deletion, of tumor antigen--specific CTLs during the progressive growth of tumors prevents costimulation by anti-4-1BB mAb. Breaking CTL ignorance by immunization with a tumor antigen-derived peptide, although insufficient to stimulate a curative CTL response, is necessary for anti--4-1BB mAb to induce a CTL response leading to the regression of established tumors. Our results suggest a new approach for immunotherapy of human cancers.

Cellular and Humoral Immunity against Vaccinia Virus Infection of Mice

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8+ T cells vs that of CD4+ T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-γ responses in CD4+ and CD8+ T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4+ T cells or B cells in IgH−/− mice, but was not sensitive to CD8+ T cell depletion alone. However, a role for CD8+ T cells in primary protection was demonstrated in MHC class II−/− mice, where depleting CD8+ T cells lead to increase severity of disease. Unlike control MHC class II−/− mice, the group depleted of CD8+ T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8+ T cells can mediate protective memory. These results collectively show that both CD4+ and CD8+ T cell-mediated immunity can contribute to protection against VV infection. However, CD4+ T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8+ T cells can contribute to protection against disease.

THE RELEVANCE OF ANIMAL MODELS IN MULTIPLE SCLEROSIS RESEARCH

Multiple Sclerosis (MS) is a complex disease with an unknown etiology and no effective cure, despite decades of extensive research that led to the development of several partially effective treatments. Researchers have only limited access to early and immunologically active MS tissue samples, and the modification of experimental circumstances is much more restricted in human studies compared to studies in animal models. For these reasons, animal models are needed to clarify the underlying immune-pathological mechanisms and test novel therapeutic and reparative approaches. It is not possible for a single mouse model to capture and adequately incorporate all clinical, radiological, pathological and genetic features of MS. The three most commonly studied major categories of animal models of MS include: (1) the purely autoimmune experimental autoimmune/allergic encephalomyelitis (EAE); (2) the virally induced chronic demyelinating disease models, with the main model of Theilers Murine Encephalomyelitis Virus (TMEV) infection and (3) toxin-induced models of demyelination, including the cuprizone model and focal demyelination induced by lyso-phosphatidyl choline (lyso-lecithine). EAE has been enormously helpful over the past several decades in our overall understanding of CNS inflammation, immune surveillance and immune-mediated tissue injury. Furthermore, EAE has directly led to the development of three approved medications for treatment in multiple sclerosis, glatiramer acetate, mitoxantrone and natalizumab. On the other hand, numerous therapeutical approaches that showed promising results in EAE turned out to be either ineffective or in some cases harmful in MS. The TMEV model features a chronic-progressive disease course that lasts for the entire lifespan in susceptible mice. Several features of MS, including the role and significance of axonal injury and repair, the partial independence of disability from demyelination, epitope spread from viral to myelin epitopes, the significance of remyelination has all been demonstrated in this model. TMEV based MS models also feature several MRI findings of the human disease. Toxin-induced demyelination models has been mainly used to study focal demyelination and remyelination. None of the three main animal models described in this review can be considered superior; rather, they are best viewed as complementary to one another. Despite their limitations, the rational utilization and application of these models to address specific research questions will remain one of the most useful tools in studies of human demyelinating diseases.

GAA Instability in Friedreich's Ataxia Shares a Common, DNA-Directed and Intraallelic Mechanism with Other Trinucleotide Diseases

We show that GAA instability in Friedreichs Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreichs Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

A human antibody that promotes remyelination enters the CNS and decreases lesion load as detected by T2-weighted spinal cord MRI in a virus-induced murine model of MS

The human monoclonal antibody rHIgM22 enhances remyelination following spinal cord demyelination in a virus‐induced murine model of multiple sclerosis. Using three‐dimensional T2‐weighted in vivo spinal cord magnetic resonance imaging (MRI), we have therefore assessed the extent of spinal cord demyelination, before and after 5 weeks of treatment with rHIgM22, to determine whether antibody enhanced remyelination can be detected by MRI. A significant decrease was seen in T2 high signal lesion volume following antibody treatment. Histologic examination of the spinal cord tissue reveals that this decrease in lesion volume correlates with antibody promoted remyelination. To show that rHIgM22 enters the spinal cord and colocalizes with demyelinating lesions, we used ultrasmall superparamagnetic iron oxide particle (USPIO)‐labeled antibodies. This may be considered as additional evidence to the hypothesis that rHIgM22 promotes remyelination by local effects in the lesions, likely by binding to CNS cells. The reduction in high signal T2‐weighted lesion volume may be an important outcome measure in future clinical trials in humans.

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theilers virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.

In vivo magnetic resonance imaging of immune cells in the central nervous system with superparamagnetic antibodies

We developed a novel MRI technique to image immune cell location and homing in vivo to the central nervous system (CNS). Superparamagnetic antibodies specific for cell surface markers allowed imaging of CD4+ T cells, CD8+ T cells, and Mac1+ cells in the CNS of mice infected with Theilers murine encephalomyelitis virus (TMEV) and in mice with experimental autoimmune encephalomyelitis (EAE). Superparamagnetic antibodies have excellent T2, T2∗, and good T1 relaxation properties, which makes them ideal MRI contrast materials. Immunohistochemistry of corresponding sections confirmed the specificity of the technique to detect immune cell types in the CNS. This powerful technique has potential to image any cell with unique surface antigens. Because superparamagnetic antibodies similar to those used in the study are approved for human use, the in vivo MRI technique we have described could be developed for human use.

Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler's virus infection

Central nervous system‐infiltrating CD8+ T cells are potential mediators of neuropathology in models of multiple sclerosis induced by Theilers murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice mount a vigorous cytotoxic T lymphocyte (CTL) response against the immunodominant virus peptide VP2121–130 and clear TMEV infection. Interferon‐γ (IFN‐γ)R‐/‐mice also mount a strong CTL response against the VP2121–130 epitope, but because of genetic deficiencies in critical IFN‐γ signaling pathways, they do not clear TMEV infection and develop prominent neurological deficits within 6 wk. This pronounced disease process, coupled with a defined CTL response, provides an ideal model for evaluating the importance of antiviral CTL activity in the development of severe demyelination and loss of motor neuron function. By administering the VP2121–130 peptide before and during TMEV infection, 99% of the VP2121–130‐specific CD8+ T cell response was inhibited. No decrease in virus infection was observed. Peptide treatment did result in significantly less motor dysfunction, even when no differences in levels of demyelination were observed. Although most investigators focus on the role of CD4+ T cells in demyelinating disease, these studies are the first to demonstrate a clear contribution of antiviral CD8+ T cells in neurological injury in a chronic‐progressive model of multiple sclerosis.

Magnetic Resonance Imaging, Microscopy, and Spectroscopy of the Central Nervous System in Experimental Animals

SummaryOver the last two decades, microscopic resolutionin vivo magnetic resonance imaging (MRI) techniques have been developed and extensively used in the study of animal models of human diseases. Standard MRI methods are frequently used in clinical studies and in the general clinical practice of human neurological diseases. This generates a need for similar studies in experimental animal research. Because small rodents are the most commonly used species as animal models of neurological diseases, the MRI techniques need to be able to provide microscopic resolution and high signal-to-noise ratio images in relatively short time. Small animal MRI systems use very high field-strength magnets, which results in higher signal to noise ratio; however, the contrast characteristics of live tissue are different at these field strengths. In addition to standard MRI techniques, several new applications have been implemented in experimental animals, including diffusion and perfusion studies, MR angiography, functional MRI studies, MRI tractography, proton and phosphorous spectroscopy, cellular and molecular imaging using novel contrast methods. Here we give an overview of how to establish a small animal imaging facility with the goal of CNS imaging. We describe the basic physical processes leading to MR signal generation, highlighting the differences between standard clinical MRI and small animal MRI. Finally, typical findings in the most common neurological disease categories and novel MRI/magnetic resonance spectroscopy methods used in their study are also described.

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide.

Theilers murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the Db gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/Db mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H‐2b haplotype mount a vigorous H‐2Db‐restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contributionof the immunodominant T cell population in resistance to TMEV, FVB/Db mice were depleted of VP2‐specific CD8+ T cells by peptide treatment prior to virus infection. Peptide‐treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the Db‐restricted CD8+ T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8+ T cells.