Lymph nodes are central sites driving effector immune responses containing highly organised secondary structures that mediated the generation of high affinity humoral and cellular immune responses. We have previously shown that stimulation of TLR4 by the vaccine adjuvant MPL (LPS) on stromal fibroblasts has an important role in regulating the timing, duration and resolution of adaptive immune responses through regulating tissue remodelling. We have shown the process is regulated by a microRNA modulating the timing of germinal centre reaction formation and lymph node structural remodelling. We have shown that an extracellular matrix product Tenascin-C also binds to TLR4 and can stimulate activation on fibroblasts and that Tenascin-C expression is regulated in lymph nodes during immune responses indicating that an endogenous natural ligand for TLR4 which may have a key role in mechanisms regulating the extent of the adaptive and innate immune responses and the organisation of lymph nodes during immune responses. The functional role of extracellular matrix in regulating immune responses and structural remodelling of secondary lymphoid tissues during an immune response are unknown.
Thus, in this project we will use a combination of vaccine and infection mediate lymph node remodelling with high-dimensional imaging, light-sheet, multi-photon live imaging and high dimensional cytometry of mice lymph nodes from wild type and mice deficient for key extracellular matrix components, focusing on Tenascin-C, to determine the role of Tenascin-C and other extracellular matrix products in regulating the extent and duration of lymph node remodelling. We will complement the imaging studies with single cell spatial genomics technologies to identify key pathways regulated during the remodelling processes in fibroblasts. To validate the capacity Tenascin-C to directly regulate processes in human fibroblasts we will use a combination of live vibrotone sections from human tonsils and primary human stromal fibroblast culture systems including 3D spheroid based “lymphoid-like” tissue bioassays developed in the Coles laboratory. This will provide a key route to translation.
TLR4 is expressed on multiple haematopoietic cell types in both human and murine the lymph nodes including macrophages and dendritic cells. The role of TLR4 ligation in myeloid lineage cell – stromal cell cross talk is not well understood. A key component of the project will be to identify how this cross talk modifies stromal and macrophage function, particularly during the resolution phase as mechanisms leading to immune resolution and restoration of non-activated lymph node architecture.
Together data from this project will provide new insights into how endogenous extracellular matrix regulating normal lymph node function, a process that is critical for vaccine function and defence to infection.
Detailed training in cellular immunology techniques, including primary cell culture, flow cytometry, and cell and tissue imaging including multi-photon confocal microscopy (in vivo tissue imaging), light sheet, confocal microscopy, high-dimensional imaging, next generation single cell RNAseq and genomics imaging. This will include training in cutting edge imaging techniques and equipment in the Kennedy Institute’s Cellular Dynamics Platform.
Training on computational image analysis including training and hands-on experience of using IMARIS, ImageJ, statistical programming language R (spatial statistics), and in high-performance computing. The student will have access to expertise in cutting edge computational software and the high-performance computational cluster KGEN.
Training in computational biology, including analysis of RNA-sequencing and single cell RNAseq and using bioinformatics pipelines. This will include both formal training and internal mentorship program in the Kennedy Institute.
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