Johnson, Aaron J.

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.

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite pre-clinical successes, therapeutic interventions have failed to extend patient survival by more than a few months. Anti-PD-1 checkpoint inhibition monotherapy has had efficacy against some tumor types but not GBM. The aim of this study was to determine whether supplementing anti-PD-1 checkpoint blockade with an engineered extended half-life IL-2 could improve outcomes in a preclinical model of disease. Our enhanced checkpoint blockade (ECB) strategy reliably cures approximately50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extended median survival even in the mice that eventually succumbed. This therapy generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. Further, many of the characteristics of brain-tumor mediated peripheral immunosuppression, including MHC class II downregulation on APCs, are prevented by ECB combination therapy. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Notably, ECB’s efficacy is independent of host MHC class I restricted antigen presentation, being equally efficacious in MHC class I and CD8 T cell deficient mice. Conversely, ECB combination therapy is reliant on CD4 T cells and their depletion abrogates the therapy’s survival benefit. Our data shows ECB combination immunotherapy to be efficacious against the GL261 glioma model through an MHC class I independent mechanism, enhancing its off-the-shelf translational appeal relative to strategies requiring extensive knowledge of tumor-specific antigens.

Abstract Glioblastoma is associated with severe and multifaceted immunosuppression affecting all immune organs. Immunosuppression in GBM is a critical barrier to the success of immunotherapies and patient survival. We demonstrated that immunosuppression in the GL261-model of experimental GBM presents with significant thymic and spleen atrophy, MHCII downregulation, presence of potent immunosuppressive factors in serum, and sequestration of T-cells in the bone marrow. Parabiosis studies determined that soluble factors mediate immunosuppression by inhibiting T-cell proliferation, thymic involution, and loss of peripheral T-cells. In contrast, bone marrow T-cell sequestration was not mediated through soluble factors. While the immunosuppression in GBM is severe, a causative link between each facet of immunosuppression and overall survival is lacking. We used two strategies to block T-cell sequestration into the bone marrow and evaluated the extent survival was impacted in experimental GBM. First, we evaluated the extent a novel and off-the-shelf combination immunotherapy that uses extended 1/2-life IL-2 and anti-PD-1 reverses bone marrow T-cell sequestration. Sham treatment or anti-PD1 monotherapy did not alter T-cell sequestration in the bone marrow and animals had no enhanced survival. Extended 1/2-life IL-2 monotherapy and combination strategy both prevented T-cell sequestration into the bone marrow. However, only combined therapy, which also prevented MHC class II downregulation, improved survival. Second, we determined that glioma-bearing adrenalectomized mice do not present with bone marrow T-cell sequestration. However, sera of glioma-bearing adrenalectomized mice is as immunosuppressive as glioma-bearing controls. Blocking bone marrow T-cell sequestration in the presences of serum immunosuppression led to no survival benefit in glioma-bearing adrenalectomized mice compared to controls. In short, bone marrow T-cell sequestration alone does not correspond with overall survival in experimental glioma. Importantly, a concerted effort to reverse MHC class II downregulation and define inhibitory circulating factors may have the highest impact in immunotherapeutic efficacy and improving patient survival.

Respiratory tissue resident–like CD8 + T cells are associated with chronic lung sequelae post-acute COVID-19.