New animal models to probe brain tumor biology, therapy, and immunotherapy: advantages and remaining concerns.

New genetic models provide better biological mimics of human tumors. The new models can give deeper insight into tumorigenesis and provide better targets for testing therapies. To use the new models most successfully, it is useful to keep in mind limitations that are harder to overcome by genetic manipulation. These include biochemical and anatomical differences between species, as well as differences in scale, both spatial and temporal. Three approaches to new genetic brain tumor models are described in the following articles. This essay provides a context, bringing out both advantages and remaining concerns. Examples are taken from work in brain tumor immunobiology and immunotherapy. The complementarity of different models, and the dichotomy between general principles and model-specific details are stressed.

Local immune regulation in the central nervous system by substance P vs. glutamate.

The response of parenchymal microglia to interferon-gamma (IFN-gamma) varies across the brain. To ask if local neurochemicals contribute to site-specific control, the influence of substance P (SP) and glutamate was evaluated in brainstem vs. hippocampus. In brainstem, stereotaxic injection of SP increased class II MHC upregulation by IFN-gamma, while a SP receptor antagonist (Spantide I) prevented it. In hippocampus, where the baseline response to IFN-gamma was lower, SP was ineffective, but blocking glutamate enhanced the response in a proportion of rats. Attempts to understand and control immune activity in the CNS should take the local neurochemical environment into account.

Local neurochemicals and site-specific immune regulation in the CNS.

Although it is often described as "immunologically privileged," the brain can display vigorous immune activity, both clinically and experimentally. The underlying control mechanisms are under active study. Here we shift attention from the brain as a whole to its diverse microenvironments. We review evidence that immune regulation in the brain is site-specific, and that local neurochemicals contribute to the site-specific control. Key points are illustrated by recent work from a rat model in which local injection of the proinflammatory cytokine, IFN-gamma, was used to modulate 2 essential aspects of the cell-mediated immune response: T cell entry from the blood, and expression of the MHC proteins that are needed to present antigen to the newly entered T cells. A growing number of neurologic disorders are known to be exacerbated by the immune/inflammatory network. Understanding the factors that influence local immune function may help explain the distribution of localized CNS damage and, more importantly, may suggest new therapeutic approaches for both desirable and unwanted responses.

Site-specific control of T cell traffic in the brain: T cell entry to brainstem vs. hippocampus after local injection of IFN-gamma.

Although it is known that neurotransmitters and neuropeptides can affect immune function in vitro, less is understood about how the neural environment affects immune function in the brain. Previously, we showed that regulation of parenchymal class II MHC after local injection of IFN-gamma is site-specific. In this companion study, we defined the effect of local IFN-gamma on the entry of class II-restricted T cells to the brain parenchyma. To activate endogenous T cells, adult CDF rats were immunized with a normal neural antigen (MBP). Two weeks later, the proinflammatory cytokine IFN-gamma (100 to 10,000 U/site) was injected stereotaxically into two neurochemically and anatomically distinct sites, the hippocampus (area CAI) and brainstem (nucleus of the solitary tract). Monoclonal R73 was used to detect T cells on cryostat sections. The greatest difference was seen 48 h after 300 U IFN-gamma was injected at each site, when there were several-fold more parenchymal T cells in the brainstem than in the hippocampus. Most parenchymal T cells were CD4+ /class II-restricted. Thus, parenchymal T cell entry and parenchymal class II up-regulation show the same hierarchy (brainstem >> hippocampus) after local IFN-gamma injection, although T cell entry was more sensitive to the IFN-gamma dose. We suggest that the local regulatory environment contributes to site-specific immune regulation, and discuss implications for the distribution of MS plaques and other aspects of local immune control. Further, in interpreting the many previous studies of cytokine-mediated immune changes in the CNS, the possibility of site-specific differences should be considered.

Site-specific immune regulation in the brain: differential modulation of major histocompatibility complex (MHC) proteins in brainstem vs. hippocampus.

Although neurotransmitters and neuropeptides are known to affect immune function in vitro and in non-neural tissues, little is known about how the local mix of neurochemicals affects immune function in the brain. Here, we study local modulation of the class II major histocompatibility complex (MHC) proteins, which present antigen to T cells in a key pathway for cell-mediated immune activity. Two sites that are well-separated anatomically and have very different neuroregulatory environments, the brainstem and hippocampus, were compared. The class II-upregulating cytokine, gamma interferon (IFN-gamma, 0.1 to 10,000 U/site), was injected stereotaxically into the hippocampus and contralateral brainstem of adult Charles-derived Fischer rats. Four days later, monoclonal antibody staining was used to detect class II MHC proteins on cryostat sections, followed by computer-assisted image analysis. As compared to hippocampus, the brainstem showed enhanced class II expression at lower IFN-gamma doses, and reached a higher plateau. Site-specific class II modulation was also seen within the layers of the hippocampus, and among other brain sites. Injection of marker protein to visualize the spread of injected protein, plus injection of IFN-gamma into alternative sites, suggested that preferential flow cannot explain all of the site-specific effects. We suggest that the local neuroregulatory environment and/or intrinsic differences among target microglia are likely to play a role. Implications for the distribution of pathological changes, such as multiple sclerosis plaques, and for local immunotherapy are discussed.

Interpreting MHC class I expression and class I/class II reciprocity in the CNS: reconciling divergent findings.

MHC-restricted T cells are thought to contribute to clinical demyelination in MS and other circumstances. The step-by-step mechanisms involved and ways of controlling them are still being defined. Identification of the MHC+ cells in the CNS in situ has been controversial. This chapter reviews MHC expression in neural tissue, including normal, pathological, experimental, and developing tissue in situ and isolated cells in vitro. A basic pattern is defined, in which MHC expression is limited to nonneural cells and strongest class I and II expression are on different cell types. Variations from the basic pattern are reviewed. Ways of reconciling divergent findings are discussed, including the use of "mock tissue" to help choose between technical and biological bases for divergent findings, the potential contribution of internal antigen to the in situ staining patterns, and the possibility that class I upregulation is actively suppressed in situ. Functional implications of the observed patterns of MHC expression and ways of confirming the function of each MHC+ cell type in situ are described. It is suggested that modulating MHC expression in different cell types at different times or in different directions might be desirable.

MHC expression in nonlymphoid tissues of the developing embryo: strongest class I or class II expression in separate populations of potential antigen-presenting cells in the skin, lung, gut, and inter-organ connective tissue.

We define expression of major histocompatibility complex (MHC) antigens in the nonlymphoid tissues of the developing rat. Antibodies to class I heavy and light chains (b2-m), and to class II MHC proteins were used. Strongest MHC expression was by individual cells in the skin, lung, gut, and inter-organ connective tissue. The class I+ and class II+ cells were distinct populations, differing in morphology, distribution, and expression of macrophage-associated antigens. A nonimmunologic role for MHC proteins in development has been proposed. Yet the distributions and antigenic profiles lead us to emphasize immunologic functions that may be served by the early presence of MHC+ cells outside the forming lymphoid organs. Potential contributions to establishment of extrathymic or maternal/fetal tolerance are discussed. Localization of strongest MHC expression to individual connective tissue cells of the developing organs, rather than parenchymal cells, is of clinical relevance to transplantation of fetal tissue.

Enhanced T cell migration to sites of microscopic CNS disease: complementary treatments evaluated by 2- and 3-D image analysis.

Novel therapies are being developed to attack tumour or other abnormal cells within the brain. A general problem is the need for delivery to sites of microscopic disease. Leukocytes offer an attractive solution; they are able to both move through tissue and recognize abnormal targets. Leukocytes may act as effectors, or as vehicles for drugs, retroviral vectors or other agents. Here, we illustrate complementary ways of enhancing leukocyte migration to sites of microscopic central nervous system (CNS) disease. Enhanced T cell migration to sites of disseminated tumour is used as the example. Computer-assisted image analysis is used to evaluate migration patterns in 2 and 3 dimensions. Shared regulatory features in the migration of tumour and responding cells, and the opportunities and questions they imply, are discussed.

Induction of HLA class I and class II expression in human T-lymphotropic virus type I-infected neuroblastoma cells.

Human T-lymphotropic virus type I (HTLV-I) is associated with a neurologic disease, HTLV-I-associated myelopathy-tropical spastic paraparesis, in which both pathological and immunological changes are observed within the central nervous system. The pathogenesis of infection in HTLV-I-associated myopathy-tropical spastic paraparesis is not well understood with respect to the cell tropism of HTLV-I and its relationship to the destruction of neural elements. In this study, neuroblastoma cells were infected with HTLV-I by coculturing with HUT-102 cells to demonstrate that cells of neuronal origin are susceptible to this retroviral infection. HTLV-I infection of the neuroblastoma cells was confirmed by verifying the presence of HTLV-I gp46 surface antigens by flow cytometry and by verifying the presence of HTLV-I pX RNA by Northern (RNA) blotting and in situ hybridization techniques. To determine whether HTLV-I infection could potentially lead to changes in cell surface recognition by the immune system, the infected neuroblastoma cells were analyzed for altered HLA expression. The HTLV-I-infected, cocultured neuroblastoma cells were shown, through cell surface antigen expression and RNA transcripts, to express HLA classes I and II. In contrast, cocultured neuroblastoma cells that did not become infected with HTLV-I expressed only HLA class I. HLA class I expression was enhanced by the cytokines tumor necrosis factor alpha and gamma interferon and in the presence of HUT-102 supernatant. In this system, expression of HLA class I and II molecules appeared to be regulated by different mechanisms. HLA class I expression was probably induced by cytokines present in the HUT-102 supernatant and was not dependent on HTLV-I infection. HLA class II expression required HTLV-I infection of the cells. The observation of HTLV-I infection leading to HLA induction in these neuroblastoma cells provides a possible mechanism for immunologic recognition of infected neuronal cells.

Tumor uptake of 99mTc-MIBI and 201Tl by a 9L gliosarcoma brain tumor model in rats.

There have been several recent case reports of the accumulation of 99mTc-MIBI [hexakismethoxyisobutylisonitriletechnetium(I), Cardiolite, Sestamibi] in tumors, but no reports of the uptake of this radiopharmaceutical in an animal model. To address this question, the biodistributions of 99mTc-MIBI and 201Tl were compared in Fisher rats bearing 9L gliosarcomas. The results showed that, although the absolute uptake of the tracers by the tumor is relatively low (< 1% ID/g), the tumor-to-normal brain ratios are greater than 6:1 because of low uptake by normal brain. The tumor-to-normal brain ratio of 99mTc-MIBI exceeds that of other currently available 99mTc radiopharmaceuticals suggesting that 99mTc-MIBI may be of particular value in the clinical evaluation of brain tumors and that further investigation of this class of compounds as tumor-avid radiopharmaceuticals is necessary.

Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes.

MHC class I molecules are coexpressed with beta 2-microglobulin (beta 2-M) on many somatic cells. However, these proteins are normally not present on cells of the central nervous system (CNS). Cells derived from human neuroblastomas were used as a model for investigating the molecular basis for the paucity of MHC class I and beta 2-M gene expression in neural cells and for the induction of these genes by two cytokines, IFN-gamma, and TNF-alpha. These cytokines independently increased MHC class I and beta 2-M cell surface expression on the neuroblastoma cell lines. IFN-gamma or TNF-alpha also increased MHC class I and beta 2-M steady-state RNA levels and the expression of MHC class I and beta 2-M CAT reporter constructs transiently transfected into the neuroblastoma cell lines, indicating that the cytokines acted by increasing the transcription of these genes. MHC class I and beta 2-M genes share two conserved regulatory elements, an NF kappa B-like site and the IFN consensus sequence, that act as a constitutive enhancer and an IFN-responsive element, respectively. Low MHC class I and beta 2-M gene expression in these cells was accounted for by undetectable to low factor binding activity specific for the above regulatory elements of these genes. TNF-alpha increased factor binding activity specific for the NF kappa B-like elements and IFN-gamma increased factor binding activity specific for the IFN consensus sequence elements of the MHC class I and beta 2-M genes, but not vice versa. Taken together, our results indicated that IFN-gamma and TNF-alpha increased MHC class I and beta 2-M gene expression in the neuroblastoma cell lines by inducing factor binding to the regulatory elements present in both genes.

Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ.

We extend use of the lacZ reporter gene for tumor biology. Intracerebral growth of 9L/lacZ, a gliosarcoma cell line that stably expresses lacZ, was evaluated in syngeneic rats. The reporter gene product, Escherichia coli-derived beta-galactosidase (beta-gal), was detected histochemically on tissue sections. This permits visualization of disseminated tumor and, as shown here, facilitates image analysis. We show that the beta-gal marker protein itself can serve as a tumor antigen in appropriate contexts. Quantitative image analysis of tumor areas is used to show that immunization with beta-gal protects against tumor growth. Abnormal beta-gal- areas are easily detected, facilitating study of antigenic modulation. The tumor studied did not escape through this mechanism. All abnormal beta-gal- areas examined were shown to reflect accumulation of inflammatory or reactive cells, not tumor. Taken together, these findings show several ways in which the lacZ reporter gene can be exploited to facilitate quantitative analysis of disseminated tumor growth within the brain. They draw attention to the growing appreciation that tumor antigens need not be cell surface molecules.

Disseminating tumor cells and their interactions with leukocytes visualized in the brain.

Brain tumors are increasingly prevalent. Recent advances focus attention on individual, disseminated tumor cells that cannot be imaged or eliminated. Cells of the immune system may be ideally suited to attack individual tumor cells, but more basic understanding is needed. We describe a rat model, using the lacZ reporter gene, that allows identification of individual tumor cells, and tumor-leukocyte interactions in vivo. The model demonstrates how widely tumor can disseminate, without secondary tumorigenesis or recruitment of nonneoplastic cells. It demonstrates that leukocytes have access to disseminating tumor. Among its many applications, this work lays a foundation for developing cell-mediated immunotherapy against individual brain tumor cells.

Effects of gamma-interferon on major histocompatibility complex antigen expression and lymphocytic infiltration in the 9L gliosarcoma brain tumor model: implications for strategies of immunotherapy.

Effects of gamma-interferon (IFN-gamma) on immune parameters in the 9L gliosarcoma model were examined. IFN-gamma increased class I major histocompatibility complex (MHC) expression in 9L cells in vitro. In vivo, intratumor injections of IFN-gamma led to increased numbers of inflammatory cells within the tumor and class II+ mononuclear phagocytes at its periphery, and increased MHC class I or II expression by endothelial and ependymal cells. Class I expression in 9L cells themselves was not increased. This suggests that there may be inhibition of class I induction in vivo for certain cell types, for which immunotherapies based on non-MHC restricted mechanisms may be more effective.

Immune modulation within the brain: recruitment of inflammatory cells and increased major histocompatibility antigen expression following intracerebral injection of interferon-gamma.

Intracerebral injections of interferon-gamma (IFN-gamma) have multiple immunological effects on rat brain, affecting all anatomic compartments. Lymphocytes and other inflammatory cells are recruited to the injection site: CD4+ T-cells into the perivascular space, OX42+ monocytes/macrophages into brain parenchyma. IFN-gamma also recruits OX8+ cells into brain parenchyma. These OX8+ cells are not stained by 'pan' T-cell antibodies, however, suggesting that they may be natural killer cells. IFN-gamma also causes increased major histocompatibility complex expression on brain cells: class I antigen on local endothelial and ependymal cells, and class II antigen on microglial, ependymal, and perivascular cells throughout both hemispheres of the brain.

Major histocompatibility complex antigen expression in the affected tissues in amyotrophic lateral sclerosis.

Monoclonal antibody immunocytochemistry was used to examine spinal cord and muscle in amyotrophic lateral sclerosis for changes that would indicate ongoing or potential immune activity. Increased expression of class I and II major histocompatibility complex (MHC) antigens was seen in the affected areas of spinal cord. New MHC expression was concentrated in phagocytes, particularly in degenerating white matter in which they were dispersed in the tissue and also packed around blood vessels. MHC antigen was not revealed in motor neurons or skeletal muscle fibers. An anti-pan-T-cell monoclonal revealed small numbers of T cells in degenerating white matter. Similar changes have been seen in other neurodegenerative disorders. They suggest a potential for (secondary) cell-mediated activity in the affected areas rather than an ongoing MHC-restricted T-cell response. Vessel-associated phagocytes may be a source of antigen to peripheral lymphoid tissue, stimulating production of the autoantibodies that have been described.

Specific uptake of the auger electron-emitting thymidine analogue 5-[123I/125I]iodo-2'-deoxyuridine in rat brain tumors: diagnostic and therapeutic implications in humans.

Glial neoplasms of the human central nervous system are malignancies that have defied treatment. Part of the problem lies in the limitations of current diagnostic techniques which are unable to identify small collections of neoplastic glia within normal parenchyma and in the difficulty of sterilizing these tumors because of limited selectivity of the cytotoxic agents available. The thymidine analogue 5-iodo-2'-deoxyuridine (IdUrd) radiolabeled with 123I and 125I was injected directly into an intracerebral rat 9L gliosarcoma and found to be a sensitive and specific agent for the detection of this neoplasm in rats. External gamma camera imaging (123I) visualized tumors as small as 0.5 mm in diameter. Autoradiography (125I) indicated that IdUrd was incorporated into the DNA of neoplastic glia only. Since 123I emits gamma-photons suitable for scintigraphy, [123I]IdUrd holds promise for the diagnosis of brain tumors in humans as well. Furthermore, since 123I and 125I are Auger electron emitters that have demonstrated antineoplastic effects, direct administration of [123I]IdUrd or [125I]IdUrd into tumors may also have potential for the treatment of central nervous system malignancies.

The effects of irradiation on major histocompatibility complex expression and lymphocytic infiltration in the normal rat brain and the 9L gliosarcoma brain tumor model.

The effects of irradiation on major histocompatibility complex (MHC) expression and lymphocytic infiltration in the normal rat brain and the 9L gliosarcoma brain tumor model were examined. Doses of irradiation administered were biologically equivalent to that used in the treatment of patients with malignant gliomas. No significant change in immune parameters was observed following irradiation in the normal rat brain. In the 9L gliosarcoma model irradiation did not suppress MHC expression or lymphocytic infiltration. These findings suggest that prior exposure to therapeutic irradiation need not adversely affect subsequent immunotherapies, and provide a foundation for future studies of immunomodulation in the irradiated brain.