Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Mayo Clinic, Rochester

Blood--brain barrier (BBB) disruption is an integral feature of numerous neurological disorders. However, there is a relative lack of knowledge regarding the underlying molecular mechanisms of immune-mediated BBB disruption. We have previously shown that CD8 T cells and perforin play critical roles in initiating altered permeability of the BBB in the peptide-induced fatal syndrome (PIFS) model developed by our laboratory. Additionally, despite having indistinguishable CD8 T cell responses, C57BL/6J (B6) mice are highly susceptible to PIFS, exhibiting functional motor deficits, increased astrocyte activation, and severe CNS vascular permeability, while 129S1/SvImJ (129S1) mice remain resistant. Therefore, to investigate the potential role of genetic factors, we performed a comprehensive genetic analysis of (B6 x 129S1) F2 progeny to define quantitative trait loci (QTL) linked to the phenotypic characteristics stated above that mediate CD8 T cell-initiated BBB disruption.

Quantitative trait loci analysis reveals candidate genes implicated in regulating functional deficit and CNS vascular permeability in CD8 T cell-initiated blood--brain barrier disruption.

The addition of antiangiogenic therapy to the standard-of-care treatment regimen for recurring glioblastoma has provided some clinical benefits while also delineating numerous caveats, prompting evaluation of the elicited alterations to the tumor microenvironment. Of critical importance, given the steadily increasing incorporation of immunotherapeutic approaches clinically, is an enhanced understanding of the interplay between angiogenic and immune response pathways within tumors. In the present study, the GL261 glioma mouse model was used to determine the effects of antiangiogenic treatment in an immune-competent host. Following weekly systemic administration of aflibercept, an inhibitor of vascular endothelial growth factor, tumor volume was assessed by magnetic resonance imaging and changes to the tumor microenvironment were determined. Treatment with aflibercept resulted in reduced tumor burden and increased survival compared with controls. Additionally, decreased vascular permeability and preservation of the integrity of tight junction proteins were observed. Treated tumors also displayed hallmarks of anti-angiogenic evasion, including marked upregulation of vascular endothelial growth factor expression and increased tumor invasiveness. Aflibercept was then administered in combination with a picornavirus-based antitumor vaccine and tumor progression was evaluated. This combination therapy significantly delayed tumor progression and extended survival beyond that observed for either therapy alone. As such, this work demonstrates the efficacy of combined antiangiogenic and immunotherapy approaches for treating established gliomas and provides a foundation for further evaluation of the effects of antiangiogenic therapy in the context of endogenous or vaccine-induced inflammatory responses.

Improved Treatment Efficacy of Antiangiogenic Therapy when Combined with Picornavirus Vaccination in the GL261 Glioma Model.

CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population.

Modulatory effects of perforin gene dosage on pathogen-associated blood-brain barrier (BBB) disruption.

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45 cells showed greater percentage of CD44CD62 activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant D:VP2 and engineered K:OVA epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the D class I molecule. FVB/D and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy  T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/D mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/D mice elicited a strong CD8 T-cell response toward the immunodominant D-restricted TMEV-derived peptide, VP2, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2D class I molecule.

Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2D class I molecule.

Human cerebral malaria (HCM) is a serious complication of  infection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale magnetic resonance imaging (MRI) study in humans identified brain swelling as the most prominent predictor of fatal HCM. Therefore, in this study, we sought to define the mechanism controlling brain edema through the use of the murine experimental cerebral malaria (ECM) model. Specifically, we investigated the ability of CD8 T cells to initiate brain edema during ECM. We determined that areas of blood-brain barrier (BBB) permeability colocalized with a reduction of the cerebral endothelial cell tight-junction proteins claudin-5 and occludin. Furthermore, through small-animal MRI, we analyzed edema and vascular leakage. Using gadolinium-enhanced T1-weighted MRI, we determined that vascular permeability is not homogeneous but rather confined to specific regions of the brain. Our findings show that BBB permeability was localized within the brainstem, olfactory bulb, and lateral ventricle. Concurrently with the initiation of vascular permeability, T2-weighted MRI revealed edema and brain swelling. Importantly, ablation of the cytolytic effector molecule perforin fully protected against vascular permeability and edema. Furthermore, perforin production specifically by CD8 T cells was required to cause fatal edema during ECM. We propose that CD8 T cells initiate BBB breakdown through perforin-mediated disruption of tight junctions. In turn, leakage from the vasculature into the parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes to ECM pathology.

Perforin Expression by CD8 T Cells Is Sufficient To Cause Fatal Brain Edema during Experimental Cerebral Malaria.

Perforin is critical for controlling viral infection and tumor surveillance. Clinically, mutations in perforin are viewed as unfavorable, as lack of this pore-forming protein results in lethal, childhood disease, familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). However, many mutations in the coding region of  are not yet associated with disease. Animal models of viral-associated blood-brain barrier (BBB) disruption and experimental cerebral malaria (ECM) have identified perforin as critical for inducing pathologic central nervous system CNS vascular permeability. This review focuses on the role of perforin in both protecting and promoting human disease. It concludes with a novel hypothesis that diversity observed in the  gene may be an example of selective advantage that protects an individual from perforin-mediated pathology, such as BBB disruption.

Finding a Balance between Protection and Pathology: The Dual Role of Perforin in Human Disease.

The contribution of antigen-presenting cell (APC) types in generating CD8 T cell responses in the central nervous system (CNS) is not fully defined, limiting the development of vaccines and understanding of immune-mediated neuropathology. Here, we generate a transgenic mouse that enables cell-specific deletion of the H-2Kb MHC class I molecule. By deleting H-2K on dendritic cells and macrophages, we compare the effect of each APC in three distinct models of neuroinflammation: picornavirus infection, experimental cerebral malaria, and a syngeneic glioma. Dendritic cells and macrophages both activate CD8 T cell responses in response to these CNS immunological challenges. However, the extent to which each of these APCs contributes to CD8 T cell priming varies. These findings reveal distinct functions for dendritic cells and macrophages in generating CD8 T cell responses to neurological disease.

Non-equivalent antigen presenting capabilities of dendritic cells and macrophages in generating brain-infiltrating CD8  T cell responses.

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2D The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2D LoxP-transgenic mouse system using otherwise MHC class I-deficient C57BL/6 mice, thereby conditionally ablating MHC class I-restricted Ag presentation in targeted APC subpopulations. We observed that CD11c APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2 Loss of H-2D on CD11c APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2D-deficient LysM APCs retained early priming of D:VP2 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2D-restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2, with functional implications both for T cell-mediated viral control and immunopathology.

Conditional Silencing of H-2D Class I Molecule Expression Modulates the Protective and Pathogenic Kinetics of Virus-Antigen-Specific CD8 T Cell Responses during Theiler's Virus Infection.

Aaron J. Johnson

Department of Immunology

Mayo Clinic, Rochester

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Aaron J. Johnson

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Mayo Clinic, Rochester

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Department of Immunology