Johnson, Aaron J.

Abstract Cerebral malaria (CM) is a severe complication arising from Plasmodium falciparum infection which results in high mortality. Lack of mechanistic insight into this pathology has resulted in ineffective therapeutic strategies. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM) our lab recapitulated hallmarks of human CM using small animal imaging, including blood-brain barrier (BBB) permeability, severe edema and microhemorrhage visible via T1 and T2-weighted MRI. Previously it had been demonstrated that infiltration of CD8 T cells with cytotoxic capacity is required to induce BBB permeability and ECM. The observation that not only infiltration, but cytotoxic killing is required provoked us to analyze differential MHC class I-restricted priming among discrete antigen presenting cell (APC) types. Using new MHC class I conditional knockout mice generated by our team, we addressed CD8 T cell priming and capacity to disrupt the BBB in ECM. Our findings revealed that mice lacking the H-2Kb MHC class I molecule expression in CD11c+ cells, but not LysM+ cells, were protected from the development of lethal ECM. Subsequently, conditional ablation of the H-2Db on these same subsets yielded similar results. Therefore, we demonstrate that both Db and Kb class I molecule expression by CD11c+ cells enable CD8 T cell priming that results in BBB permeability during ECM. In contrast, CD8 T cells primed by H-2Kb or H-2Db expressing LysM+ APCs are not capable of inducing BBB disruption. These results imply discrete APC subsets prime functionally distinct CD8 T cell responses in the CNS and modulate the outcome of cerebral malaria. This work begins to bridge the mechanistic gap and will impact future therapeutic strategies.

Abstract Glioblastoma multiforme (GBM) is incurable and among the most lethal of cancers. Immunotherapy for GBM is hampered by the fact that GBM patients are severely immunosuppressed with dangerously low T cell counts. Importantly, immunotherapy for GBM will be ineffective without defining the mechanism of immunosuppression. Given that GBM does not metastasize, we investigated the possibility of neuro-immune interactions using the GL261 glioma model in C57BL/6 mice. C57BL/6 mice harboring GL261 gliomas have abnormal thymuses which are acutely and significantly involuted compared to healthy control mice. Glioma bearing mice also have significant thymic atrophy which increases with tumor burden. Further structural analysis of the thymus in glioma bearing mice revealed disrupted thymic epithelial cells and reduced EPCAM+ cell expression of MHC II. Thymic subsets in tumor bearing mice were also developmentally skewed with increased generation of single positive T cells and a block at double negative 2 (DN2) to DN3 transition. Peripherally, MHCII expression was also significantly decreased in blood derived cells in glioma bearing mice. This correlated with an overall reduction in total CD4 T cell numbers. The results of this study demonstrate the GL261 model is recapitulating the immune suppression observed in GBM patients. These results also support the presence of a brain-thymus interaction that could negatively impact the development of immunotherapy strategies to treat GBM.

Abstract One of the most severe complications of Plasmodium falciparum infection in humans is cerebral malaria. Cerebral malaria causes coma, neurological deficits, and potentially death. The disease is characterized by blood-brain barrier (BBB) disruption leading to vascular permeability. In this study, we investigated cerebral endothelial cell tight junction protein alterations and BBB breakdown in the Plasmodium berghei ANKA(PbA) model of experimental cerebral malaria. PbAinfected C57BL/6 mice displayed significant areas of vascular leakage within the central nervous system at six days post infection. Regions of permeability co-localized with disruption of the BBB tight junction proteins claudin-5 and occludin on microvasculature as shown using confocal microscopy. Additionally, 3D MRI analysis revealed that PbA infected mice exhibited vascular leakage specific to the hypothalamus, thalamus, and brain stem. In contrast, PbA infected perforin deficient mice retained tight junction integrity and displayed significantly reduced vascular leakage. We propose that CD8 T cells initiate vascular permeability through disruption of cerebral endothelial cell tight junctions. In turn, the resulting imbalance of homeostasis in vital brain regions likely contributes to the pathology and morbidity associated with PbA infection.

Abstract MHC multimers including tetramers and pentamers provide a powerful way to visualize antigen specific T cell responses in both experimental and clinical immune assays. The multimers can be acquired commercially, from the NIH, or assembled in the laboratory for measuring immune responses to commonly-studied or novel-hypothetical antigens. The multimer reagents can be expensive and time consuming to acquire and in many cases are uncontrolled reagents as robust positive control T cells used to assess the reagents functionally may not be available. This is particularly problematic when using MHC multimers to investigate less well-defined T cell responses, such as those characterized by low T cell frequencies or by specificity to hypothetical antigens relevant to cancer, infection, or autoimmune disease. These problems are magnified when studying irreplaceable clinical trial samples where having confidence that reagents are biochemically intact is critical. To address this problem, we have devised a versatile, flow cytometry-based, capture assay to probe the structural integrity of MHC multimer reagents. In principle, the idea is to immobilize on a bead antibody specific for conformational determinants expressed on a properly folded MHC molecule. The particle becomes labeled and can be visualized and quantified by flow cytometry when a fluorescently tagged tetramer is adsorbed to the particulate upon binding to the antibody.

Abstract The most severe clinical complication of Plasmodium falciparum infection is cerebral malaria (CM). Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice is an established model of CM. Previous work has implied a role for CD8 T cells and perforin expression in fatal stages of PbA infection. We therefore infected C57BL/6 and C57BL/6 Perforin-/- mice with PbA and assessed the CNS for cerebral endothelial cell blood-brain barrier (BBB) disruption using FITC albumin leakage into tissue, confocal microscopy and 3D volumetric T2 and T1 gadolinium enhanced MRI. PbA infected C57BL/6 mice: 1.) had extensive disruption of BBB tight junction proteins Occludin and Claudin 5, 2.) developed severe CNS vascular permeability of FITC albumin and gadolinium leakage, and 3.) became moribund. We also observed increased VEGF cytokine expression coinciding with CNS vascular permeability in these animals. Despite having similar CD8 T cell infiltration into the brain, PbA infected C57BL/6 Perforin-/- mice had intact BBB tight junction, were devoid of CNS vascular permeability, presented with normal brain MRI, and had normal behavioral scores on the rotarod assay. This study is therefore the first to demonstrate the critical importance of perforin expression in promoting BBB tight junction alteration during PbA infection. Therefore, development of therapies to block factors related perforin mediated delivery could have significant benefit to the treatment of CM.

Abstract Blood brain barrier (BBB) disruption is a common feature of neurological disease. We developed a novel model of CD8 T cell mediated BBB disruption using the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis. At 7 days post TMEV infection, 50-70% of CNS infiltrating CD8 T cells are specific for an immunodominant virus peptide VP2 121-130 presented in the context of the Db class I molecule. Intravenous injection of VP2 121-130 peptide at 7 days post TMEV infection results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. Despite having an identical CD8 T cell response, 129 SvIm mice survive administration of the VP2 121-130 peptide. We performed bone marrow transplants to determine if a hematopoetic factor was responsible for this fatal syndrome. 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Similar brain infiltration and purity of transplanted CD8 T cells was observed during acute TMEV infection in reconstituted mice. However, 129 SvIm mice reconstituted with C57BL/6 bone marrow had increased microhemorrhage formation as measured by T2* weighted MRI and became moribund. Meanwhile, 129 SvIm mice receiving an autologous bone marrow transplant had significantly less microhemorrhage formation and remained resistant to becoming moribund. These studies demonstrate that a hematopoetic factor is responsible for the onset of brain microhemorrhage in immune mediated fatal BBB disruption.

Abstract INTRODUCTION: Plasma extracellular vesicles (EVs) have been shown as a promising source for biomarker identification in glioblastoma (GBM) and could help differential diagnosis, treatment evaluation and tumor progression monitoring. These EVs are enriched in molecular signatures indicative of their cell origins, giving an indication of the key players in this pathology. In this project, we aimed to identify diagnostic biomarkers for GBM plasma EVs and their cells of origin. METHODS: Plasma EV samples were prepared following the MIFlowCyt-EV guideline of the International Society for Extracellular Vesicles, then stained for EV markers (CD9/CD63/CD81) and markers indicative of cell origins (CD31/CD45/CD41a/CD11b). Actin phalloidin was used as a negative marker. Stained plasma samples were analyzed using a Cytek Aurora flow cytometer. Percentages of different EV subpopulations were analyzed and compared between GBM and normal donor (ND) plasma EVs (reference group). Further clustering analysis was performed on EV events by t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) analysis. The predictive value of multiparametric qualities derived from the reference group was tested in blinded test group samples. RESULTS: Percentages of CD9, CD81, and CD11b positive EVs were higher in GBM patient plasma, while ND plasma had more CD41a positive EVs. GBM plasma EVs had unique multiparametric signatures compared to ND plasma EVs based on t-SNE and FlowSOM analysis. Our analysis also identified 15 distinct EV subpopulations which differed in size and various surface marker expression levels. Eight of these subpopulations were enriched for GBM EVs, while three were enriched for ND EVs. Our method of multiparametric analysis demonstrates high sensitivity and specificity in predicting disease status in human samples. CONCLUSIONS: GBM plasma EVs have a unique surface marker expression profile and distinct EV subpopulations compared to ND plasma EVs. Multiparametric signatures show promise as potential diagnostic markers of GBM.

SARS-CoV-2 mRNA vaccination induces robust humoral and cellular immunity in the circulation; however, it is currently unknown whether it elicits effective immune responses in the respiratory tract, particularly against variants of concern (VOCs), including Omicron. We compared the SARS-CoV-2 S–specific total and neutralizing antibody responses, and B and T cell immunity, in the bronchoalveolar lavage fluid (BAL) and blood of COVID-19–vaccinated individuals and hospitalized patients. Vaccinated individuals had significantly lower levels of neutralizing antibody against D614G, Delta (B.1.617.2), and Omicron BA.1.1 in the BAL compared with COVID-19 convalescents despite robust S-specific antibody responses in the blood. Furthermore, mRNA vaccination induced circulating S-specific B and T cell immunity, but in contrast to COVID-19 convalescents, these responses were absent in the BAL of vaccinated individuals. Using a mouse immunization model, we demonstrated that systemic mRNA vaccination alone induced weak respiratory mucosal neutralizing antibody responses, especially against SARS-CoV-2 Omicron BA.1.1 in mice; however, a combination of systemic mRNA vaccination plus mucosal adenovirus-S immunization induced strong neutralizing antibody responses not only against the ancestral virus but also the Omicron BA.1.1 variant. Together, our study supports the contention that the current COVID-19 vaccines are highly effective against severe disease development, likely through recruiting circulating B and T cell responses during reinfection, but offer limited protection against breakthrough infection, especially by the Omicron sublineage. Hence, mucosal booster vaccination is needed to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2, including infection by the Omicron sublineage and future VOCs.

2038 Background: Glioblastoma (GBM) is the most common malignant brain tumor with poor clinical prognosis. Management of GBM is hampered by the lack of an accurate test that can be used for differential diagnosis of tumor progression from inflammatory pseudoprogression. Plasma extracellular vesicles (EVs) has been shown to be a promising source for biomarker identification. In this project, we aimed to identify GBM plasma EV markers that could serve as the basis of a liquid biopsy. Methods: Sample preparation, assay controls and instrument calibration were performed following MIFlowCyt-EV guideline. Plasma samples were subjected to 2-step centrifugations to remove cell debris and platelets. 10ul of plasma sample was diluted with 90ul filtered PBS, then stained for EV surface markers including CD9, CD31, CD45, CD41a and CD11b, as well as actin phalloidin. Stained plasma samples were purified using IZON qEV1/70nm column, then EV fractions were analyzed using full spectrum Cytek Aurora flow cytometer. Clustering analysis was performed on EV events (CD9 +/ actin phalloidin -) using t-SNE and FlowSOM extensions from FlowJo plugins. Results: Compared to normal donors, GBM plasma EVs were bigger in size (higher SSC value) and expressed higher levels of CD9, CD31, CD45 and CD11b while ND plasma EVs had higher CD41a expression. t-SNE and FlowSOM analysis demonstrated that GBM plasma EVs had a unique surface marker expression profile compared to ND EVs. It also showed 10 EV sub-populations that differed in size as well as various surface marker expression levels. Four of these subpopulations were enriched in GBM EVs, while three of these were enriched in ND EVs. Conclusions: This multiparametric analysis revealed that GBM plasma EVs had a unique surface marker expression profile compared to ND plasma EVs. Further separation and molecular profiling analysis based on each sub population could reveal EV biomarkers that are unique to each sample population.