LMP student seminars: 7 October

Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.

Anyone is welcome. No need to register.

Location: Medical Sciences Building, rooms 4171 or 4279, see below.

As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.

Group 3: Cardiovascular, Physiology and Metabolism

Location: MSB 4171

Tara Lazetic

• Title: TBA
• Supervisor: TBA

Catherine Lin

• Title: Utilizing Magnetic Resonance-Guided Focused Ultrasound To Enhance Delivery And Efficacy of Immune-Based Therapeutics in Diffuse Intrinsic Pontine Glioma
• Supervisor: Dr. James Rutka

Group 5: Infectious Diseases, Inflammation and Immunology

Location: MSB 4279

Yijia (Kelly) Liu 

• Title: Single-Cell Atlas of Chronic Woodchuck Hepatitis-induced HCC Highlights Conserved Tumor-Driven Immune Exhaustion Shared with Human HCC
• Supervisor: Dr. Sonya MacParland

Kathryn Lloyd-Smith

• Title: Flagellins induce an alternative TLR5-independent pathway in human intestinal organoids
• Supervisor: Dr. Stephen Girardin

Abstracts

Tara Lazetic: TBA

TBA

Catherine Lin: Utilizing Magnetic Resonance-Guided Focused Ultrasound To Enhance Delivery And Efficacy of Immune-Based Therapeutics in Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG) is a brain tumour that arises in the pons and remains the leading cause of brain tumour-related deaths in children. A major obstacle to treatment is the lack of targeted therapies and limited drug delivery across the intact blood-brain barrier (BBB). Techniques such as magnetic resonance-guided focused ultrasound (MRgFUS) can transiently open the BBB and have recently been shown to modulate immune responses. I hypothesize that MRgFUS can enhance the efficacy of immune-based therapies for DIPG by improving delivery and augmenting anti-tumour immune activity. Immunocompetent DIPG mouse models are generated through the RCAS-Tva system or stereotactic injection of a murine DIPG cell line. BBB integrity of the murine models is characterized using magnetic-resonance imaging (MRI). Immunohistochemistry and imaging mass cytometry are conducted on tumour-bearing brain tissue to visualize the tumour-immune microenvironment. Non-tumour bearing mice are used to optimize MRgFUS parameters for BBB opening. A controller system, in which the acoustic pressure of the focused ultrasound automatically adjusts based on the acoustic feedback received, is used for improved safety during sonication. Results show that the BBB is intact in the DIPG allograft model but not the RCAS-Tva model. Increased macrophage infiltration was observed at the tumour site compared to non-tumour bearing controls, and all immunocompetent DIPG mouse models characterized to date express targetable antigens or immune checkpoints, including GD2, TIM3, and B7-H3. Ongoing studies focus on optimizing delivery of chimeric-antigen T cells using MRgFUS.

Yijia (Kelly) Liu: Single-Cell Atlas of Chronic Woodchuck Hepatitis-induced HCC Highlights Conserved Tumor-Driven Immune Exhaustion Shared with Human HCC

Background: The response rate of immune-checkpoint inhibitors in chronic hepatitis B (CHB)-induced HCC patients remains below 40%. Such low rate might be attributed to a limited cellular-level understanding of the HCC niche. Thus, a systematic characterization of the HCC ecosystem across paired, spatially distinct HCC-related regions is crucial for identifying major contributors to immune dysfunction.

Methods: Single-cell RNA sequencing was performed on peripheral blood mononuclear cells (PBMCs) and liver biopsies from healthy (n=8) and woodchuck hepatitis virus (WHV)-induced HCC woodchucks (n=9). Sampling from HCC animals included matched non-malignant liver tissue (n=8), HCC margins (n=20), and multiple intratumoral regions (n=23). The transcriptomic analysis was coupled with histology to visualize changes in liver morphology.

Results: An integrated single-cell map of 275,220 cells was generated from a total of 74 samples, capturing 5 major immune cell lineages shared with human HCC. T cell profiling within HCC animals revealed a significant enrichment of exhausted CD8+ T cells in HCC margin and tumor compared to non-malignant tumor-distal tissues (p<0.01 and p<0.05), characterized by an increase in the expression of exhaustion-related markers and a decrease in the expression of cytotoxicity-related markers. Coinciding with a higher CD8+ T cell exhaustion score, WHV-induced HCC margin and tumor were enriched in immunoregulatory myeloid lineages with high expression of angiogenic and immunomodulatory mediators. Analysis of cell-cell interaction using CellChat revealed multiple regulatory signaling pathways between tumor-enriched myeloid cells and CD8+ T cells that might promote T cell dysfunction.

Conclusion: Our single-cell characterization of WHV-induced woodchuck HCC demonstrated a transcriptionally and morphologically proliferative tumor subtype and revealed an immune-exhausted tumor landscape that mirrors human HCC. Future in vitro validations on woodchuck myeloid-T cell interaction will aim to dissect the mechanistic role of myeloid-derived factors in modulating T cell exhaustion, which will reveal therapeutic targets that can be directly tested using WHV-induced woodchuck HCC.

Kathryn Lloyd-Smith: Flagellins induce an alternative TLR5-independent pathway in human intestinal organoids

The innate immune response is crucial in maintaining microbial homeostasis and intestinal barrier integrity. A large class of cytoplasmic pattern recognition receptors (PRRs), NOD-like receptors (NLRs), recognize danger- or microbial-associated molecular patterns (DAMPs/MAMPs) and can activate inflammasomes, leading to caspase-1 activation, maturation of cytokines including IL-1β and IL-18, and pyroptotic cell death through gasdermin D (GSDMD) pore formation. However, our lab has identified an alternative pathway in human intestinal organoids involving caspase-8 and gasdermin E (GSDME) that occurs in the absence of canonical inflammasome activation or cytokine release. Flagellin, a flagellar protein found in many bacteria, is sensed both intracellularly by NLRC4 and extracellularly by Toll-like receptor 5 (TLR5). While flagellin was previously considered immunogenic, recent work has shown its ability to activate TLR5 is structure-dependent. However, there is little evidence showing whether structural differences in flagellins translate to altered NLRC4 activation. My work investigates the responses of human intestinal organoids to the three classes of flagellin: stimulator, silent, and evader. I show that select flagellins can induce cleavage of caspase-8 and GSDME despite lacking the structural components required for TLR5 activation, while caspase-1 and GSDMD cleavage remain undetected, suggesting the response is mediated through an alternative pathway consistent with NLRC4 dependence. Together, these findings demonstrate that human intestinal organoids can mount distinct responses to structurally diverse flagellins and do so through an alternative caspase-8/GSDME pathway.

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions.