Intravital imaging in immunology: experimental and computational approaches

Thursday, June 17 at 04:15am (PDT)
Thursday, June 17 at 12:15pm (BST)
Thursday, June 17 08:15pm (KST)

SMB2021 SMB2021 Follow Wednesday (Thursday) during the "MS18" time block.
Note: this minisymposia has multiple sessions. The second session is MS17-IMMU (click here).

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Barun Majumder (University of Tennessee, USA), Soumen Bera (University of Tennessee, USA)


Intravital Imaging with multi-photon microscopy is one of the most powerful tools to answer some of the longstanding fundamental questions of cell biology, tumor biology and immunology. New and emerging technologies in the field of intravital imaging have helped scientists study cell dynamics and interactions at very high resolutions in tissues in vivo. Based on the labeling and detection of a particular wavelength (fluorescence), the technique can provide insights on cell’s cardinal attributes like division, localization, migration and interaction in a molecular level in three dimensions over time. There are three major steps in the intravital imaging studies: the experimental design, data extraction and processing prior to use in analysis, and the data analysis and mathematical modeling. In this proposed mini-symposium we will invite experts in the area of experimental immunology and mathematical modeling to discuss the challenges, advancements, limitations and accomplishments made in the field in the context of immunology and cell biology.

Paulus Mrass

(Department of Molecular Genetics and Microbiology, University of New Mexico, USA)
"Quantitative imaging identifies CXCR4 as a molecular switch that balances confinement and ballisitic migration of cytotoxic T cells within flu- infected lungs"
Cytotoxic T cells play an important role in protective immune responses against the flu, but the molecular mechanisms that regulate this function remain incompletely understood. In the present study we established a live imaging model that enables quantification of T cell motility within intact flu-infected lung tissue. This setup revealed that cytotoxic T cells show heterogenous migration patterns, characterized by intermittent periods of confinement and ballistic relocation. A special feature of our imaging model was the capacity to separately measure T cells that are in close proximity to flu-infected regions and those that are distant. Comparison of these two groups revealed that T cells that reside in flu-positive regions are signficantly more confined than T cells in flu-negative regions. This finding indicated that exposure to cognate peptides is one mechanism that contributes to the heterogeneous migration patterns of cytotoxic T cells within flu-infected lungs. To dissect the molecular mechanisms that regulate interstitial migration of T cells further, we analyzed T cell motility after treatment of lungs with pharmacological inhibitors. This approach revealed that AMD3100, a specific inhibitor of the chemokine recetpor CXCR4, caused a signficant suppression of interstitial migration within flu-negative regions. Unexpectely, we also found that inhibition of CXCR4 had an oppositive effect of T cells within flu-positive regions, i.e. the T cells became less confined. From these findings, we conclude that CXCR4 functions as a molecular switch that boosts interaction with target cells by two distinct mechanisms: (1) by enhancing motility towards flu-positive regions; and (2) by limiting motility within flu- positive regions, which likely facilitates the initiation of cognate interactions with target cells. Indeed, when we inhibited CXCR4 in flu-infected mice with AMD3100, this led to a reduction of degranulation of cytotoxic T cells infiltrating flu-infected lungs. Together, quantitative imaging has revealed that CXCR4 controls the functionality of lung-infiltrating cytotoxic T cells by regulation of intra-tissue motility.

Arja Ray

(Department of Pathology, University of California San Francisco, USA)
"Visualizing T cell behavior in solid tumors to define barriers to immunotherapy"
Cancer immunotherapy relies on the effective function of cytotoxic CD8 T cells in the tumor microenvironment (TME). Other immune cells such as tumor- associated macrophages (TAMs) and the tumor stroma are critical components of the TME that inform CD8 T cell function. In tumors with abundant T cell infiltration, immunotherapy using bi-specific T cell engagers (BiTE) mediates physical interactions between T cells and tumor cells, thereby forcing tumor recognition and cytotoxic killing. However, this immunotherapy has had limited success in solid tumors, leading to questions regarding the barriers posed by the TME in this context. Using intravital imaging, we discovered vast heterogeneity in the movement of BiTEs out of perfused blood vessels in intact live tumors, from unhindered diffusion in some regions to being entirely contained within blood vessels in others. Indeed, the sufficiency of tumor-resident T cells to mediate tumor rejection was a function of dosage, thereby indicating that the bioavailability of such functional molecules in the TME is a key factor restricting their efficacy in solid tumors. Many solid tumors, on the other hand, are characterized by a lack of T cell (and other immune cell) infiltration, commonly referred to as an “immune desert” tumor. It has been postulated that TAMs play a key role in trapping T cells at the tumor margins, thereby leading to a T cell sparse tumor nest. Using a novel mouse model to specifically mark TAMs, we performed live imaging of TAM:T cell localization and interactions in the TME. Indeed, in an immune desert tumor model, T cells tend to be trapped near the tumor margins, co-localized with TAMs on a bed of robust deposition of fibrous collagen. Using spatial transcriptomics, we identify a unique TAM population at the tumor margin that are putatively involved in fibrosis in communication with CAFs. We hypothesize that this TAM subset is a key component of the immune- stromal cross-talk that leads to excessive fibrosis and exclusion of T cells from the TME in immune desert tumors. Overall, visualizing and defining the microenvironment around T cells in immune rich and immune desert tumors reveals distinct barriers to effective T cell function and points to the necessity of tailored approaches to improve cancer immunotherapy for different solid tumors.

Judy Cannon

(University of New Mexico School of Medicine, USA)
"Effect of tissue environment on T cell movement"
T cells are a key effector cell type in the immune response, migrating through tissues in order to clear infection such as influenza infection in the lung. T cells must move through many different types of tissues to mount an effective response: naïve T cells migrate in and out of lymph nodes searching for antigen on dendritic cells, while activated T cells migrate to peripheral tissue such as lung to clear influenza infection. We investigate how different tissues such as lymph node and lung environments affect T cell motion using two photon microscopy to visualize effector T cells moving in different tissue settings. We perform quantitative analysis of in situ T cell movement and find that T cell speeds vary independent of the tissue environment or type of T cells. Naïve T cells in the lymph nodes move with similar average speed as effector T cells in the flu-infected lung, but effector T cells in an acute lung injury model move much more slowly. Interestingly, despite similar speeds, T cells in the lung do not show a coupling of speed and persistence that many other cell types have been seen to demonstrate, suggesting that the lung environment may exert effects on T cell movement to drive specific types of motion. T cells in the lung also show greater persistent motion than T cells in lymph nodes. The combination of in situ imaging and quantitative analysis of cell movement can uncover how specific tissue environments impact T cell movement and search for infection within different tissue contexts.

Soumen Bera

(Department of Microbiology, University of Tennessee Knoxville, USA)
"Mathematical modeling of CD8 T cell-mediated elimination of malaria liver stages using intravital imaging experiments"
CD8+ T cells are one of the most critical immune defenses against intracellular pathogens capable of finding and eliminating the infected cells and preventing blood-stage diseases. Intravital imaging technic helps demonstrate the killing of liver stages Malaria parasites by memory induced or activated CD8+ T cells. Using these technics and mathematical modeling, we have recently shown the formation of large clusters consisting of variable number of effectors CD8+ T cells around the parasite-infected hepatocytes is rapid, indicating the high efficiency of CD8+ T cells for finding their target within complex organs like the liver. However, it has not been clear how many activated CD8+ T cells are required to eliminate the malaria parasites within a short period of time. Using a combination of intravital experimental data and mathematical modeling, we have provided detailed insights about the CD8+ T cells dynamic against the parasite phenotypes. The parasite's death corresponding to a high number of CD8+ T cells indicates a prolonged interaction between them; however, the death of parasites with a smaller number of T cells due to multiple factors. Using alternative mechanistic models, increasing the number of CD8+ T cells response better predict the parasite phenotypic dynamics compare to others, indicating increasing CD8+ T cells prompt the killing process. However, alternative mathematical models showed the fixed killing efficiency per T cell per parasite that means a higher number of T cells has higher killing efficiency. Finally, dose-response analysis indicates a smaller number of T cells is required to kill the parasites after a couple of hours of CD8+ T cells transfer, but with increasing time, a high number of T cells is required to eliminate the parasite. With different alternative methods, our analysis indicates novel insights about quantifying CD8+ T cells dynamic in the process of parasite elimination. 

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Virtual conference of the Society for Mathematical Biology, 2021.