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The Mechanisms and Dynamics Controlling Cycles of Lymph Node Expansion

Periodic Reporting for period 3 - LNEXPANDS (The Mechanisms and Dynamics Controlling Cycles of Lymph Node Expansion )

Reporting period: 2021-03-01 to 2022-08-31

Lymph node swelling is a classical hallmark of immunity. This expansion is observed by doctors, researchers and patients, yet as obvious as this process is, our understanding of the remodelling mechanisms involved are in their infancy. Lymph node remodelling is rapid and yet completely reversible, occurring countless times throughout our lifetimes. The purpose of this proposal is to understand how lymph node remodelling occurs and is resolved, repeatedly; to understand immunity in a whole organ context.
The architecture of lymphoid organs is key to the effective operation of our immune system and is dictated by structures formed by non-haematopoetic stromal cells, including endothelial cells, and fibroblasts. Beyond their structural roles, stromal cells play an active role in immune responses, and the field of stromal immunology has become one of the most dynamic and exciting areas of immunology research. In this proposal I focus on the changing behaviour of fibroblastic reticular cells (FRCs) throughout cycles of lymph node remodelling. Fibroblastic reticular cells (FRCs) are the most abundant lymphoid stromal cell population, and form an interconnected network spanning the full volume of the tissue. FRCs are highly contractile and are able to relax and stretch during early phases of lymph node remodelling. FRCs proliferate during later phases of lymph node growth but are then removed as homeostasis is restored. Throughout this proposal I will use an extensive range of systems, ranging from proteomics and biochemistry to intravital imaging.
I aim to: 1) To discover how spreading and stretching of the existing fibroblastic reticular network is directed in the acute phase of expansion: 2) To discover the cellular cues inducing the switch from stretching to proliferation and growth of the fibroblastic reticular network and 3) understand how homeostasis is regained as immune responses are resolved.
Aim 1 - Mechanisms of acute lymph node expansion
Phospho-proteomics screen of FRCs stimulated with CLEC-2-Fc revealed a decrease in phosphorylation of the actin-binding protein MARCKS in FRCs upon short-term CLEC-2 stimulation. MARCKS is required for neuronal growth cone migration during brain development, and we hypothesised that FRCs use MARCKS to spread in similar manner, reminiscent of developing and spreading neurones.
We have generated FRCs stably expressing GFP-tagged MARCKS to investigate this hypothesis. MARCKS has a cytosolic localisation in steady-state FRCs, whilst CLEC-2 stimulation causes MARCKS to shift to the membrane, where it preferentially clusters within membrane protrusions. We now plan to move our investigations in vivo, with conditional deletion of MARCKS in FRCs during responses to immunisation.

Aim 2 - Mechanics of the fibroblastic reticular cell network
During an adaptive immune response, the number of lymphocytes in the LN rapidly increases, expanding the size of the LN. The FRC network must react to accommodate this, however how the FRCs maintain an intact network during this large change in tissue size is not well understood. We have measured the changes to the fibroblastic reticular network tension in lymphoid tissue.

Aim 3 - Understanding tissue remodelling of lymph node resolution
Following an infection, lymph nodes are able to return to their original size and architecture. However in some cases of chronic infection, homeostasis is not restored, and as a result immunity is compromised. Absolute numbers of FRCs are increased during immune responses, when the lymph node size is expanded to maximum capacity, which is generally accepted to be due to proliferation. However, when the threat is resolved, the lymph node must return to homeostasis and reduce in size. The numbers of FRCs are also reduced as lymph node size decreases, but the signals controlling this remodeling, and the mechanisms for stromal cell removal are unknown. We have undertaken extensive flow cytometry and CyTOF analysis to determine the kinetics of lymph node resolution.
We have generated MARCKS KO cell lines and shown that MARCKS is required for FRCs to respond to dendritic cells in vitro.

Laser ablation experiments ex-vivo have shown that network tension increases during early phases of lymph node expansion and we are now investigating whether this increased network tension is a physical cue to stimulate proliferation of the fibroblast network.

We have generated a new mouse model (PDGFRa-mGFP- CreERT2) as proposed to enable the conditional and specific deletion or expression of genes with the fibroblastic reticular cells, during resolution phase. We have validated that this model when crossed to PDPNfl/fl mice can selectively and efficiently delete podoplanin from FRCs in vivo.

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